Methods compositions, and kits comprising linker probes for quantifying polynucleotides

ABSTRACT

The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/575,661, filed May 28, 2004, for “Methods, Compositions, and Kits forQuantifying Target Polynucleotides” by Chen and Zhou.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 6, 2009, is named533USC1.txt, and is 118,670 bytes in size.

FIELD

The present teachings are in the field of molecular and cell biology,specifically in the field of detecting target polynucleotides such asmiRNA.

INTRODUCTION

RNA interference (RNAi) is a highly coordinated, sequence-specificmechanism involved in posttranscriptional gene regulation. During theinitial steps of process, a ribonuclease (RNase) II-like enzyme calledDicer reduces long double-strand RNA (dsRNA) and complex hairpinprecursors into: 1) small interfering RNAs (siRNA) that degrademessenger RNA (mRNA) and 2) micro RNAs (miRNAs) that can target mRNAsfor cleavage or attenuate translation.

The siRNA class of molecules is thought to be comprised of 21-23nucleotide (nt) duplexes with characteristic dinucleotide 3′ overhangs(Ambros et al., 2003, RNA, 9 (3), 277-279). siRNA has been shown to actas the functional intermediate in RNAi, specifically directing cleavageof complementary mRNA targets in a process that is commonly regarded tobe an antiviral cellular defense mechanism (Elbashir et al., 2001,Nature, 411:6836), 494-498, Elbashir et al., 2001, Genes andDevelopment, 15 (2), 188-200). Target RNA cleavage is catalyzed by theRNA-induced silencing complex (RISC), which functions as a siRNAdirected endonuclease (reviewed in Bartel, 2004, Cell, 116 (2),281-297).

Micro RNAs (miRNAs) typically comprise single-stranded, endogenousoligoribonucleotides of roughly 22 (18-25) bases in length that areprocessed from larger stem-looped precursor RNAs. The first genesrecognized to encode miRNAs, lin-4 and let-7 of C. elegans, wereidentified on the basis of the developmental timing defects associatedwith the loss-of-function mutations (Lee et al., 1993, Cell, 75 (5),843-854; Reinhart et al., 2000, Nature, 403, (6772), 901-906; reviewedby Pasquinelli et al., 2002, Annual Review of Cell and DevelopmentalBiology, 18, 495-513). The breadth and importance of miRNA-directed generegulation are coming into focus as more miRNAs and regulatory targetsand functions are discovered. To date, a total of at least 700 miRNAshave been identified in C. elegans, Drosophila (Fire et al., 1998,Nature, 391 (6669(, 805-811), mouse, human (Lagos-Quintana et al., 2001,Science, 294 (5543), 853-858), and plants (Reinhart et al., 2002, Genesand Development, 16 (13), 1616-1626). Their sequences are typicallyconserved among different species. Size ranges from 18 to 25 nucleotidesfor miRNAs are the most commonly observed to date.

The function of most miRNAs is not known. Recently discovered miRNAfunctions include control of cell proliferation, cell death, and fatmetabolism in flies (Brennecke et al., 2003, cell, 113 (1), 25-36; Xu etal, 2003, Current Biology, 13 (9), 790-795), neuronal patterning innematodes (Johnston and Hobert, 2003, Nature, 426 (6968), 845-849),modulation of hematopoietic lineage differentiation in mammals (Chen etal., 2004, Science, 303 (5654), 83-87), and control of leaf and flowerdevelopment in plants (Aukerman and Sakai, 2003, Plant Cell, 15 (11),2730-2741; Chen, 2003, Science, 303 (5666):2022-2025; Emery et al.,2003, Current Biology, 13 (20), 1768-1774; Palatnik et al., 2003,Nature, 425 (6955), 257-263). There is speculation that miRNAs mayrepresent a new aspect of gene regulation.

Most miRNAs have been discovered by cloning. There are few cloning kitsavailable for researchers from Ambion and QIAGEN etc. The process islaborious and less accurate. Further, there has been little reliabletechnology available for miRNA quantitation (Allawi et al., Third WaveTechnologies, RNA. 2004 July; 10(7):1153-61). Northern blotting has beenused but results are not quantitative (Lagos-Quitana et al., 2001,Science, 294 (5543), 853-854). Many miRNA researchers are interested inmonitoring the level of the miRNAs at different tissues, at thedifferent stages of development, or after treatment with variouschemical agents. However, the short length of miRNAs has their studydifficult.

SUMMARY

In some embodiments, the present teachings provide a method fordetecting a micro RNA (miRNA) comprising; hybridizing the miRNA and alinker probe, wherein the linker probe comprises a stem, a loop, and a3′ target-specific portion, wherein the 3′ target-specific portion basepairs with the 3′ end region of the miRNA; extending the linker probe toform an extension reaction product; amplifying the extension reactionproduct to form an amplification product; and, detecting the miRNA.

In some embodiments, the present teachings provide a method fordetecting a target polynucleotide comprising; hybridizing the targetpolynucleotide and a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′target-specific portion base pairs with the 3′ end region of the targetpolynucleotide; extending the linker probe to form an extension reactionproduct; amplifying the extension reaction product to form anamplification product in the presence of a detector probe, wherein thedetector probe comprises a nucleotide of the linker probe stem in theamplification product or a nucleotide of the linker probe stemcomplement in the amplification product; and, detecting the targetpolynucleotide.

In some embodiments, the present teachings provide a method fordetecting a miRNA molecule comprising; hybridizing the miRNA moleculeand a linker probe, wherein the linker probe comprises a stem, a loop,and a 3′ target specific portion, wherein the 3′ target-specific portionbase pairs with the 3′ end region of the target polynucleotide;extending the linker probe to form an extension reaction product;amplifying the extension reaction product in the presence of a detectorprobe to form an amplification product, wherein the detector probecomprises a nucleotide of the linker probe stem in the amplificationproduct or a nucleotide of the linker probe stem complement in theamplification product, and the detector probe further comprises anucleotide of the 3′ end region of the miRNA in the amplificationproduct or a nucleotide of the 3′ end region of the miRNA complement inthe amplification product; and, detecting the miRNA molecule.

In some embodiments, the present teachings provide a method fordetecting two different miRNAs from a single hybridization reactioncomprising; hybridizing a first miRNA and a first linker probe, and asecond miRNA and a second linker probe, wherein the first linker probeand the second linker probe each comprise a loop, a stem, and a 3′target-specific portion, wherein the 3′ target-specific portion of thefirst linker probe base pairs with the 3′ end region of the first miRNA,and wherein the 3′ target-specific portion of the second linker probebase pairs with the 3′ end region of the second miRNA; extending thefirst linker probe and the second linker probe to form extensionreaction products; dividing the extension reaction products into a firstamplification reaction to form a first amplification reaction product,and a second amplification reaction to form a second amplificationreaction product, wherein a primer in the first amplification reactioncorresponds with the first miRNA and not the second miRNA, and a primerin the second amplification reaction corresponds with the second miRNAand not the first miRNA, wherein a first detector probe in the firstamplification reaction differs from a second detector probe in thesecond amplification reaction, wherein the first detector probecomprises a nucleotide of the first linker probe stem of theamplification product or a nucleotide of the first linker probe stemcomplement in the first amplification product, wherein the seconddetector probe comprises a nucleotide of the second linker probe stem ofthe amplification product or a nucleotide of the second linker probestem complement in the amplification product; and, detecting the twodifferent miRNAs.

In some embodiments, the present teachings provide a method fordetecting two different target polynucleotides from a singlehybridization reaction comprising; hybridizing a first targetpolynucleotide and a first linker probe, and a second targetpolynucleotide and a second linker probe, wherein the first linker probeand the second linker probe each comprise a loop, a stem, and a 3′target-specific portion, wherein the 3′ target-specific portion of thefirst linker probe base pairs with the 3′ end region of the first targetpolynucleotide, and wherein the 3′ target-specific portion of the secondlinker probe base pairs with the 3′ end region of the second targetpolynucleotide; extending the first linker probe and the second linkerprobe to form extension reaction products; dividing the extensionreaction products into a first amplification reaction to form a firstamplification reaction product and a second amplification reaction toform a second amplification reaction product; and, detecting the twodifferent miRNA molecules.

In some embodiments, the present teachings provide a method fordetecting a miRNA molecule from a cell lysate comprising; hybridizingthe miRNA molecule from the cell lysate with a linker probe, wherein thelinker probe comprises a stem, a loop, and a 3′ target specific portion,wherein the 3′ target-specific portion base pairs with the 3′ end regionof the miRNA; extending the linker probe to form an extension reactionproduct; amplifying the extension reaction product to form anamplification product in the presence of a detector probe, wherein thedetector probe comprises a nucleotide of the linker probe stem of theamplification product or a nucleotide of the linker probe stemcomplement in the amplification product, and the detector probe furthercomprises a nucleotide of the 3′ end region of the miRNA in theamplification product or a nucleotide of the 3′ end region of the miRNAcomplement in the amplification product; and, detecting the miRNAmolecule.

A kit comprising; a reverse transcriptase and a linker probe, whereinthe linker probe comprises a stern, a loop, and a 3′ target-specificportion, wherein the 3′ target-specific portion corresponds to a miRNA.

The present teachings contemplate method for detecting a miRNA moleculecomprising a step of hybridizing, a step of extending, a step ofamplifying, and a step of detecting.

These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 depicts certain aspects of various compositions according to someembodiments of the present teachings.

FIG. 2 depicts certain aspects of various compositions according to someembodiments of the present teachings.

FIG. 3 depicts certain sequences of various compositions according tosome embodiments of the present teachings.

FIG. 4 depicts one single-plex assay design according to someembodiments of the present teachings.

FIG. 5 depicts an overview of a multiplex assay design according to someembodiments of the present teachings.

FIG. 6 depicts a multiplex assay design according to some embodiments ofthe present teachings.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way. The section headings usedherein are for organizational purposes only and are not to be construedas limiting the described subject matter in any way. All literature andsimilar materials cited in this application, including but not limitedto, patents, patent applications, articles, books, treatises, andinternet web pages are expressly incorporated by reference in theirentirety for any purpose. When definitions of terms in incorporatedreferences appear to differ from the definitions provided in the presentteachings, the definition provided in the present teachings shallcontrol. It will be appreciated that there is an implied “about” priorto the temperatures, concentrations, times, etc discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein. In this application, the use ofthe singular includes the plural unless specifically stated otherwise.For example, “a primer” means that more than one primer can, but neednot, be present; for example but without limitation, one or more copiesof a particular primer species, as well as one or more versions of aparticular primer type, for example but not limited to, a multiplicityof different forward primers. Also, the use of “comprise”, “comprises”,“comprising”, “contain”, “contains”, “containing”, “include”,“includes”, and “including” are not intended to be limiting. It is to beunderstood that both the foregoing general description and the followingdetailed description are exemplary and explanatory only and are notrestrictive of the invention.

Some Definitions

As used herein, the term “target polynucleotide” refers to apolynucleotide sequence that is sought to be detected. The targetpolynucleotide can be obtained from any source, and can comprise anynumber of different compositional components. For example, the targetcan be nucleic acid (e.g. DNA or RNA), transfer RNA, siRNA, and cancomprise nucleic acid analogs or other nucleic acid mimic. The targetcan be methylated, non-methylated, or both. The target can bebisulfite-treated and non-methylated cytosines converted to uracil.Further, it will be appreciated that “target polynucleotide” can referto the target polynucleotide itself, as well as surrogates thereof, forexample amplification products, and native sequences. In someembodiments, the target polynucleotide is a miRNA molecule. In someembodiments, the target polynucleotide lacks a poly-A tail. In someembodiments, the target polynucleotide is a short DNA molecule derivedfrom a degraded source, such as can be found in for example but notlimited to forensics samples (see for example Butler, 2001, Forensic DNATyping: Biology and Technology Behind STR Markers. The targetpolynucleotides of the present teachings can be derived from any of anumber of sources, including without limitation, viruses, prokaryotes,eukaryotes, for example but not limited to plants, fungi, and animals.These sources may include, but are not limited to, whole blood, a tissuebiopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen,biowarfare agents, anal secretions, vaginal secretions, perspiration,saliva, buccal swabs, various environmental samples (for example,agricultural, water, and soil), research samples generally, purifiedsamples generally, cultured cells, and lysed cells. It will beappreciated that target polynucleotides can be isolated from samplesusing any of a variety of procedures known in the art, for example theApplied Biosystems ABI Prism™ 6100 Nucleic Acid PrepStation, and the ABIPrism™ 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat.No. 5,234,809, mirVana RNA isolation kit (Ambion), etc. It will beappreciated that target polynucleotides can be cut or sheared prior toanalysis, including the use of such procedures as mechanical force,sonication, restriction endonuclease cleavage, or any method known inthe art. In general, the target polynucleotides of the present teachingswill be single stranded, though in some embodiments the targetpolynucleotide can be double stranded, and a single strand can resultfrom denaturation.

As used herein, the term “3′ end region of the target polynucleotide”refers to the region of the target to which the 3′ target specificportion of the linker probe hybridizes. In some embodiments there can bea gap between the 3′ end region of the target polynucleotide and the 5′end of the linker probe, with extension reactions filling in the gap,though generally such scenarios are not preferred because of the likelydestabilizing effects on the duplex. In some embodiments, a miRNAmolecule is the target, in which case the term “3′ end region of themiRNA” is used.

As used herein, the term “linker probe” refers to a molecule comprisinga 3′ target specific portion, a stem, and a loop. Illustrative linkerprobes are depicted in FIG. 2 and elsewhere in the present teachings. Itwill be appreciated that the linker probes, as well as the primers ofthe present teachings, can be comprised of ribonucleotides,deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, modified phosphate-sugar-backboneoligonucleotides, nucleotide analogs, or combinations thereof. For someillustrative teachings of various nucleotide analogs etc, see Fasman,1989, Practical Handbook of Biochemistry and Molecular Biology, pp.385-394, CRC Press, Boca Raton, Fla., Loakes, N. A. R. 2001, vol29:2437-2447, and Pellestor et al., Int J Mol Med. 2004 April;13(4):521-5.), references cited therein, and recent articles citingthese reviews. It will be appreciated that the selection of the linkerprobes to query a given target polynucleotide sequence, and theselection of which collection of target polynucleotide sequences toquery in a given reaction with which collection of linker probes, willinvolve procedures generally known in the art, and can involve the useof algorithms to select for those sequences with minimal secondary andtertiary structure, those targets with minimal sequence redundancy withother regions of the genome, those target regions with desirablethermodynamic characteristics, and other parameters desirable for thecontext at hand.

As used herein, the term “3′ target-specific portion” refers to thesingle stranded portion of a linker probe that is complementary to atarget polynucleotide. The 3′ target-specific portion is locateddownstream from the stem of the linker probe. Generally, the 3′target-specific portion is between 6 and 8 nucleotides long. In someembodiments, the 3′ target-specific portion is 7 nucleotides long. Itwill be appreciated that routine experimentation can produce otherlengths, and that 3′ target-specific portions that are longer than 8nucleotides or shorter than 6 nucleotides are also contemplated by thepresent teachings. Generally, the 3′-most nucleotides of the 3′target-specific portion should have minimal complementarity overlap, orno overlap at all, with the 3′ nucleotides of the forward primer; itwill be appreciated that overlap in these regions can produce undesiredprimer dimer amplification products in subsequent amplificationreactions. In some embodiments, the overlap between the 3′-mostnucleotides of the 3′ target-specific portion and the 3′ nucleotides ofthe forward primer is 0, 1, 2, or 3 nucleotides. In some embodiments,greater than 3 nucleotides can be complementary between the 3′-mostnucleotides of the 3′ target-specific portion and the 3′ nucleotides ofthe forward primer, but generally such scenarios will be accompanied byadditional non-complementary nucleotides interspersed therein. In someembodiments, modified bases such as LNA can be used in the 3′ targetspecific portion to increase the Tm of the linker probe (see for examplePetersen et al., Trends in Biochemistry (2003), 21:2:74-81). In someembodiments, universal bases can be used, for example to allow forsmaller libraries of linker probes. Universal bases can also be used inthe 3′ target specific portion to allow for the detection of unknowntargets. For some descriptions of universal bases, see for exampleLoakes et al., Nucleic Acids Research, 2001, Volume 29, No. 12,2437-2447. In some embodiments, modifications including but not limitedto LNAs and universal bases can improve reverse transcriptionspecificity and potentially enhance detection specificity.

As used herein, the term “stem” refers to the double stranded region ofthe linker probe that is between the 3′ target-specific portion and theloop. Generally, the stem is between 6 and 20 nucleotides long (that is,6-20 complementary pairs of nucleotides, for a total of 12-40 distinctnucleotides). In some embodiments, the stem is 8-14 nucleotides long. Asa general matter, in those embodiments in which a portion of thedetector probe is encoded in the stem, the stem can be longer. In thoseembodiments in which a portion of the detector probe is not encoded inthe stem, the stem can be shorter. Those in the art will appreciate thatstems shorter that 6 nucleotides and longer than 20 nucleotides can beidentified in the course of routine methodology and without undueexperimentation, and that such shorter and longer stems are contemplatedby the present teachings. In some embodiments, the stem can comprise anidentifying portion.

As used herein, the term “loop” refers to a region of the linker probethat is located between the two complementary strands of the stern, asdepicted in FIG. 1 and elsewhere in the present teachings. Typically,the loop comprises single stranded nucleotides, though other moietiesmodified DNA or RNA, Carbon spacers such as C18, and/or PEG(polyethylene glycol) are also possible. Generally, the loop is between4 and 20 nucleotides long. In some embodiments, the loop is between 14and 18 nucleotides long. In some embodiments, the loop is 16 nucleotideslong. As a general matter, in those embodiments in which a reverseprimer is encoded in the loop, the loop can generally be longer. Inthose embodiments in which the reverse primer corresponds to both thetarget polynucleotide as well as the loop, the loop can generally beshorter. Those in the art will appreciate that loops shorter that 4nucleotides and longer than 20 nucleotides can be identified in thecourse of routine methodology and without undue experimentation, andthat such shorter and longer loops are contemplated by the presentteachings. In some embodiments, the loop can comprise an identifyingportion.

As used herein, the term “identifying portion” refers to a moiety ormoieties that can be used to identify a particular linker probe species,and as a result determine a target polynucleotide sequence, and canrefer to a variety of distinguishable moieties including zipcodes, aknown number of nucleobases, and combinations thereof. In someembodiments, an identifying portion, or an identifying portioncomplement, can hybridize to a detector probe, thereby allowingdetection of a target polynucleotide sequence in a decoding reaction.The terms “identifying portion complement” typically refers to at leastone oligonucleotide that comprises at least one sequence of nucleobasesthat are at least substantially complementary to and hybridize withtheir corresponding identifying portion. In some embodiments,identifying portion complements serve as capture moieties for attachingat least one identifier portion:element complex to at least onesubstrate; serve as “pull-out” sequences for bulk separation procedures;or both as capture moieties and as pull-out sequences (see for exampleO'Neil, et al., U.S. Pat. Nos. 6,638,760, 6,514,699, 6,146,511, and6,124,092). Typically, identifying portions and their correspondingidentifying portion complements are selected to minimize: internal,self-hybridization; cross-hybridization with different identifyingportion species, nucleotide sequences in a reaction composition,including but not limited to gDNA, different species of identifyingportion complements, or target-specific portions of probes, and thelike; but should be amenable to facile hybridization between theidentifying portion and its corresponding identifying portioncomplement. Identifying portion sequences and identifying portioncomplement sequences can be selected by any suitable method, for examplebut not limited to, computer algorithms such as described in PCTPublication Nos. WO 96/12014 and WO 96/41011 and in European PublicationNo. EP 799,897; and the algorithm and parameters of SantaLucia (Proc.Natl. Acad. Sci. 95:1460-65 (1998)). Descriptions of identifyingportions can be found in, among other places, U.S. Pat. No. 6,309,829(referred to as “tag segment” therein); U.S. Pat. No. 6,451,525(referred to as “tag segment” therein); U.S. Pat. No. 6,309,829(referred to as “tag segment” therein); U.S. Pat. No. 5,981,176(referred to as “grid oligonucleotides” therein); U.S. Pat. No.5,935,793 (referred to as “identifier tags” therein); and PCTPublication No. WO 01/92579 (referred to as “addressablesupport-specific sequences” therein). In some embodiments, the stem ofthe linker probe, the loop of the linker probe, or combinations thereofcan comprise an identifying portion, and the detector probe canhybridize to the corresponding identifying portion. In some embodiments,the detector probe can hybridize to both the identifying portion as wellas sequence corresponding to the target polynucleotide. In someembodiments, at least two identifying portion: identifying portioncomplement duplexes have melting temperatures that fall within a ΔT_(m)range (T_(max)−T_(min)) of no more than 10° C. of each other. In someembodiments, at least two identifying portion: identifying portioncomplement duplexes have melting temperatures that fall within a ΔT_(m)range of 5° C. or less of each other. In some embodiments, at least twoidentifying portion: identifying portion complement duplexes havemelting temperatures that fall within a ΔT_(m) range of 2° C. or less ofeach other. In some embodiments, at least one identifying portion or atleast one identifying portion complement is used to separate the elementto which it is bound from at least one component of a ligation reactioncomposition, a digestion reaction composition, an amplified ligationreaction composition, or the like. In some embodiments, identifyingportions are used to attach at least one ligation product, at least oneligation product surrogate, or combinations thereof, to at least onesubstrate. In some embodiments, at least one ligation product, at leastone ligation product surrogate, or combinations thereof, comprise thesame identifying portion. Examples of separation approaches include butare not limited to, separating a multiplicity of different element:identifying portion species using the same identifying portioncomplement, tethering a multiplicity of different element: identifyingportion species to a substrate comprising the same identifying portioncomplement, or both. In some embodiments, at least one identifyingportion complement comprises at least one label, at least one mobilitymodifier, at least one label binding portion, or combinations thereof.In some embodiments, at least one identifying portion complement isannealed to at least one corresponding identifying portion and,subsequently, at least part of that identifying portion complement isreleased and detected, see for example Published P.C.T. ApplicationWO04/4634 to Rosenblum et al., and Published P.C.T. ApplicationWO01/92579 to Wenz et al.

As used herein, the term “extension reaction” refers to an elongationreaction in which the 3′ target specific portion of a linker probe isextended to form an extension reaction product comprising a strandcomplementary to the target polynucleotide. In some embodiments, thetarget polynucleotide is a miRNA molecule and the extension reaction isa reverse transcription reaction comprising a reverse transcriptase. Insome embodiments, the extension reaction is a reverse transcriptionreaction comprising a polymerase derived from a Eubacteria. In someembodiments, the extension reaction can comprise rTth polymerase, forexample as commercially available from Applied Biosystems catalog numberN808-0192, and N808-0098. In some embodiments, the target polynucleotideis a miRNA or other RNA molecule, and as such it will be appreciatedthat the use of polymerases that also comprise reverse transcriptionproperties can allow for some embodiments of the present teachings tocomprise a first reverse transcription reaction followed thereafter byan amplification reaction, thereby allowing for the consolidation of tworeactions in essentially a single reaction. In some embodiments, thetarget polynucleotide is a short DNA molecule and the extension reactioncomprises a polymerase and results in the synthesis of a 2^(nd) strandof DNA. In some embodiments, the consolidation of the extension reactionand a subsequent amplification reaction is further contemplated by thepresent teachings.

As used herein, the term “primer portion” refers to a region of apolynucleotide sequence that can serve directly, or by virtue of itscomplement, as the template upon which a primer can anneal for any of avariety of primer nucleotide extension reactions known in the art (forexample, PCR). It will be appreciated by those of skill in the art thatwhen two primer portions are present on a single polynucleotide, theorientation of the two primer portions is generally different. Forexample, one PCR primer can directly hybridize to a first primerportion, while the other PCR primer can hybridize to the complement ofthe second primer portion. In addition, “universal” primers and primerportions as used herein are generally chosen to be as unique as possiblegiven the particular assays and host genomes to ensure specificity ofthe assay.

As used herein, the term “forward primer” refers to a primer thatcomprises an extension reaction product portion and a tail portion. Theextension reaction product portion of the forward primer hybridizes tothe extension reaction product. Generally, the extension reactionproduct portion of the forward primer is between 9 and 19 nucleotides inlength. In some embodiments, the extension reaction product portion ofthe forward primer is 16 nucleotides. The tail portion is locatedupstream from the extension reaction product portion, and is notcomplementary with the extension reaction product; after a round ofamplification however, the tail portion can hybridize to complementarysequence of amplification products. Generally, the tail portion of theforward primer is between 5-8 nucleotides long. In some embodiments, thetail portion of the forward primer is 6 nucleotides long. Those in theart will appreciate that forward primer tail portion lengths shorterthan 5 nucleotides and longer than 8 nucleotides can be identified inthe course of routine methodology and without undue experimentation, andthat such shorter and longer forward primer tail portion lengths arecontemplated by the present teachings. Further, those in the art willappreciate that lengths of the extension reaction product portion of theforward primer shorter than 9 nucleotides in length and longer than 19nucleotides in length can be identified in the course of routinemethodology and without undue experimentation, and that such shorter andlonger extension reaction product portion of forward primers arecontemplated by the present teachings.

As used herein, the term “reverse primer” refers to a primer that whenextended forms a strand complementary to the target polynucleotide. Insome embodiments, the reverse primer corresponds with a region of theloop of the linker probe. Following the extension reaction, the forwardprimer can be extended to form a second strand product. The reverseprimer hybridizes with this second strand product, and can be extendedto continue the amplification reaction. In some embodiments, the reverseprimer corresponds with a region of the loop of the linker probe, aregion of the stem of the linker probe, a region of the targetpolynucleotide, or combinations thereof. Generally, the reverse primeris between 13-16 nucleotides long. In some embodiments the reverseprimer is 14 nucleotides long. In some embodiments, the reverse primercan further comprise a non-complementary tail region, though such a tailis not required. In some embodiments, the reverse primer is a “universalreverse primer,” which indicates that the sequence of the reverse primercan be used in a plurality of different reactions querying differenttarget polynucleotides, but that the reverse primer nonetheless is thesame sequence.

The term “upstream” as used herein takes on its customary meaning inmolecular biology, and refers to the location of a region of apolynucleotide that is on the 5′ side of a “downstream” region.Correspondingly, the term “downstream” refers to the location of aregion of a polynucleotide that is on the 3′ side of an “upstream”region.

As used herein, the term “hybridization” refers to the complementarybase-pairing interaction of one nucleic acid with another nucleic acidthat results in formation of a duplex, triplex, or other higher-orderedstructure, and is used herein interchangeably with “annealing.”Typically, the primary interaction is base specific, e.g., A/T and G/C,by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking andhydrophobic interactions can also contribute to duplex stability.Conditions for hybridizing detector probes and primers to complementaryand substantially complementary target sequences are well known, e.g.,as described in Nucleic Acid Hybridization, A Practical Approach, B.Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985) and J.Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968). In general,whether such annealing takes place is influenced by, among other things,the length of the polynucleotides and the complementary, the pH, thetemperature, the presence of mono- and divalent cations, the proportionof G and C nucleotides in the hybridizing region, the viscosity of themedium, and the presence of denaturants. Such variables influence thetime required for hybridization. Thus, the preferred annealingconditions will depend upon the particular application. Such conditions,however, can be routinely determined by the person of ordinary skill inthe art without undue experimentation. It will be appreciated thatcomplementarity need not be perfect; there can be a small number of basepair mismatches that will minimally interfere with hybridization betweenthe target sequence and the single stranded nucleic acids of the presentteachings. However, if the number of base pair mismatches is so greatthat no hybridization can occur under minimally stringent conditionsthen the sequence is generally not a complementary target sequence.Thus, complementarity herein is meant that the probes or primers aresufficiently complementary to the target sequence to hybridize under theselected reaction conditions to achieve the ends of the presentteachings.

As used herein, the term “amplifying” refers to any means by which atleast a part of a target polynucleotide, target polynucleotidesurrogate, or combinations thereof, is reproduced, typically in atemplate-dependent manner, including without limitation, a broad rangeof techniques for amplifying nucleic acid sequences, either linearly orexponentially. Exemplary means for performing an amplifying step includeligase chain reaction (LCR), ligase detection reaction (LDR), ligationfollowed by Q-replicase amplification, PCR, primer extension, stranddisplacement amplification (SDA), hyperbranched strand displacementamplification, multiple displacement amplification (MDA), nucleic acidstrand-based amplification (NASBA), two-step multiplexed amplifications,rolling circle amplification (RCA) and the like, including multiplexversions or combinations thereof, for example but not limited to,OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (alsoknown as combined chain reaction—CCR), and the like. Descriptions ofsuch techniques can be found in, among other places, Sambrook et al.Molecular Cloning, 3^(rd) Edition,; Ausbel et al.; PCR Primer: ALaboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); TheElectronic Protocol Book, Chang Bioscience (2002), Msuih et al., J.Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R.Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., CurrOpin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. No. 6,027,998; U.S.Pat. No. 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenzet al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1):152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis etal., PCR Protocols: A Guide to Methods and Applications, Academic Press(1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenauet al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin,Development of a Multiplex Ligation Detection Reaction DNA Typing Assay,Sixth International Symposium on Human Identification, 1995 (availableon the world wide web at:promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit InstructionManual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc.Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res.25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999);Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany andGelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96(1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., GenomeRes. 2003 February; 13(2):294-307, and Landegren et al., Science241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November;2(6):542-8, Cook et al., J Microbiol Methods. 2003 May; 53(2):165-74,Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12(1):21-7, U.S.Pat. Nos. 5,830,711, 6,027,889, 5,686,243, Published P.C.T. ApplicationWO0056927A3, and Published P.C.T. Application WO9803673A1. In someembodiments, newly-formed nucleic acid duplexes are not initiallydenatured, but are used in their double-stranded form in one or moresubsequent steps. An extension reaction is an amplifying technique thatcomprises elongating a linker probe that is annealed to a template inthe 5′ to 3′ direction using an amplifying means such as a polymeraseand/or reverse transcriptase. According to some embodiments, withappropriate buffers, salts, pH, temperature, and nucleotidetriphosphates, including analogs thereof, i.e., under appropriateconditions, a polymerase incorporates nucleotides complementary to thetemplate strand starting at the 3′-end of an annealed linker probe, togenerate a complementary strand. In some embodiments, the polymeraseused for extension lacks or substantially lacks 5′ exonuclease activity.In some embodiments of the present teachings, unconventional nucleotidebases can be introduced into the amplification reaction products and theproducts treated by enzymatic (e.g., glycosylases) and/orphysical-chemical means in order to render the product incapable ofacting as a template for subsequent amplifications. In some embodiments,uracil can be included as a nucleobase in the reaction mixture, therebyallowing for subsequent reactions to decontaminate carryover of previousuracil-containing products by the use of uracil-N-glycosylase (see forexample Published P.C.T. Application WO9201814A2). In some embodimentsof the present teachings, any of a variety of techniques can be employedprior to amplification in order to facilitate amplification success, asdescribed for example in Radstrom et al., Mol Biotechnol. 2004 February;26(2):133-46. In some embodiments, amplification can be achieved in aself-contained integrated approach comprising sample preparation anddetection, as described for example in U.S. Pat. Nos. 6,153,425 and6,649,378. Reversibly modified enzymes, for example but not limited tothose described in U.S. Pat. No. 5,773,258, are also within the scope ofthe disclosed teachings. The present teachings also contemplate variousuracil-based decontamination strategies, wherein for example uracil canbe incorporated into an amplification reaction, and subsequentcarry-over products removed with various glycosylase treatments (see forexample U.S. Pat. No. 5,536,649, and U.S. Provisional Application60/584,682 to Andersen et al.,). Those in the art will understand thatany protein with the desired enzymatic activity can be used in thedisclosed methods and kits. Descriptions of DNA polymerases, includingreverse transcriptases, uracil N-glycosylase, and the like, can be foundin, among other places, Twyman, Advanced Molecular Biology, BIOSScientific Publishers, 1999; Enzyme Resource Guide, rev. 092298,Promega, 1998; Sambrook and Russell; Sambrook et al.; Lehninger; PCR:The Basics; and Ausbel et al.

As used herein, the term “detector probe” refers to a molecule used inan amplification reaction, typically for quantitative or real-time PCRanalysis, as well as end-point analysis. Such detector probes can beused to monitor the amplification of the target polynucleotide. In someembodiments, detector probes present in an amplification reaction aresuitable for monitoring the amount of amplicon(s) produced as a functionof time. Such detector probes include, but are not limited to, the5′-exonuclease assay (TaqMan® probes described herein (see also U.S.Pat. No. 5,538,848) various stem-loop molecular beacons (see e.g., U.S.Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, NatureBiotechnology 14:303-308), stemless or linear beacons (see, e.g., WO99/21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001,SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097),Sunrise®/Amplifluor® probes (U.S. Pat. No. 6,548,250), stem-loop andduplex Scorpion™ probes (Solinas et al., 2001, Nucleic Acids Research29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No.6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons(U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences),hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA)light-up probes, self-assembled nanoparticle probes, andferrocene-modified probes described, for example, in U.S. Pat. No.6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al.,1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, MolecularCell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35;Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002,Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, NucleicAcids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332;Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al.,2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res.Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc14:11155-11161. Detector probes can also comprise quenchers, includingwithout limitation black hole quenchers (Biosearch), Iowa Black (IDT),QSY quencher (Molecular Probes), and Dabsyl and Dabcelsulfonate/carboxylate Quenchers (Epoch). Detector probes can alsocomprise two probes, wherein for example a fluor is on one probe, and aquencher is on the other probe, wherein hybridization of the two probestogether on a target quenches the signal, or wherein hybridization onthe target alters the signal signature via a change in fluorescence.Detector probes can also comprise sulfonate derivatives of fluoresceindyes with SO3 instead of the carboxylate group, phosphoramidite forms offluorescein, phosphoramidite forms of CY 5 (commercially available forexample from Amersham). In some embodiments, intercalating labels areused such as ethidium bromide, SYBR® Green I (Molecular Probes), andPicoGreen® (Molecular Probes), thereby allowing visualization inreal-time, or end point, of an amplification product in the absence of adetector probe. In some embodiments, real-time visualization cancomprise both an intercalating detector probe and a sequence-baseddetector probe can be employed. In some embodiments, the detector probeis at least partially quenched when not hybridized to a complementarysequence in the amplification reaction, and is at least partiallyunquenched when hybridized to a complementary sequence in theamplification reaction. In some embodiments, the detector probes of thepresent teachings have a Tm of 63-69 C, though it will be appreciatedthat guided by the present teachings routine experimentation can resultin detector probes with other Tms. In some embodiments, probes canfurther comprise various modifications such as a minor groove binder(see for example U.S. Pat. No. 6,486,308) to further provide desirablethermodynamic characteristics. In some embodiments, detector probes cancorrespond to identifying portions or identifying portion complements.

The term “corresponding” as used herein refers to a specificrelationship between the elements to which the term refers. Somenon-limiting examples of corresponding include: a linker probe cancorrespond with a target polynucleotide, and vice versa. A forwardprimer can correspond with a target polynucleotide, and vice versa. Alinker probe can correspond with a forward primer for a given targetpolynucleotide, and vice versa. The 3′ target-specific portion of thelinker probe can correspond with the 3′ region of a targetpolynucleotide, and vice versa. A detector probe can correspond with aparticular region of a target polynucleotide and vice versa. A detectorprobe can correspond with a particular identifying portion and viceversa. In some cases, the corresponding elements can be complementary.In some cases, the corresponding elements are not complementary to eachother, but one element can be complementary to the complement of anotherelement.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “reaction vessel” generally refers to anycontainer in which a reaction can occur in accordance with the presentteachings. In some embodiments, a reaction vessel can be an eppendorftube, and other containers of the sort in common practice in modernmolecular biology laboratories. In some embodiments, a reaction vesselcan be a well in microtitre plate, a spot on a glass slide, or a well inan Applied Biosystems TaqMan Low Density Array for gene expression(formerly MicroCard™). For example, a plurality of reaction vessels canreside on the same support. In some embodiments, lab-on-a-chip likedevices, available for example from Caliper and Fluidgm, can provide forreaction vessels. In some embodiments, various microfluidic approachesas described in U.S. Provisional Application 60/545,674 to Wenz et al.,can be employed. It will be recognized that a variety of reaction vesselare available in the art and within the scope of the present teachings.

As used herein, the term “detection” refers to any of a variety of waysof determining the presence and/or quantity and/or identity of a targetpolynucleotide. In some embodiments employing a donor moiety and signalmoiety, one may use certain energy-transfer fluorescent dyes. Certainnonlimiting exemplary pairs of donors (donor moieties) and acceptors(signal moieties) are illustrated, e.g., in U.S. Pat. Nos. 5,863,727;5,800,996; and 5,945,526. Use of some combinations of a donor and anacceptor have been called FRET (Fluorescent Resonance Energy Transfer).In some embodiments, fluorophores that can be used as signaling probesinclude, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5(Cy 5), fluorescein, Vic™, Liz™, Tamra™, 5-Fam™, 6-Fam™, and Texas Red(Molecular Probes). (Vic™, Liz™, Tamra™, 5-Fam™, and 6-Fam™ (allavailable from Applied Biosystems, Foster City, Calif.). In someembodiments, the amount of detector probe that gives a fluorescentsignal in response to an excited light typically relates to the amountof nucleic acid produced in the amplification reaction. Thus, in someembodiments, the amount of fluorescent signal is related to the amountof product created in the amplification reaction. In such embodiments,one can therefore measure the amount of amplification product bymeasuring the intensity of the fluorescent signal from the fluorescentindicator. According to some embodiments, one can employ an internalstandard to quantify the amplification product indicated by thefluorescent signal. See, e.g., U.S. Pat. No. 5,736,333. Devices havebeen developed that can perform a thermal cycling reaction withcompositions containing a fluorescent indicator, emit a light beam of aspecified wavelength, read the intensity of the fluorescent dye, anddisplay the intensity of fluorescence after each cycle. Devicescomprising a thermal cycler, light beam emitter, and a fluorescentsignal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907;6,015,674; and 6,174,670, and include, but are not limited to the ABIPrism® 7700 Sequence Detection System (Applied Biosystems, Foster City,Calif.), the ABI GeneAmp® 5700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.), the ABI GeneAmp® 7300 SequenceDetection System (Applied Biosystems, Foster City, Calif.), and the ABIGeneAmp® 7500 Sequence Detection System (Applied Biosystems). In someembodiments, each of these functions can be performed by separatedevices. For example, if one employs a Q-beta replicase reaction foramplification, the reaction may not take place in a thermal cycler, butcould include a light beam emitted at a specific wavelength, detectionof the fluorescent signal, and calculation and display of the amount ofamplification product. In some embodiments, combined thermal cycling andfluorescence detecting devices can be used for precise quantification oftarget nucleic acid sequences in samples. In some embodiments,fluorescent signals can be detected and displayed during and/or afterone or more thermal cycles, thus permitting monitoring of amplificationproducts as the reactions occur in “real time.” In some embodiments, onecan use the amount of amplification product and number of amplificationcycles to calculate how much of the target nucleic acid sequence was inthe sample prior to amplification. In some embodiments, one could simplymonitor the amount of amplification product after a predetermined numberof cycles sufficient to indicate the presence of the target nucleic acidsequence in the sample. One skilled in the art can easily determine, forany given sample type, primer sequence, and reaction condition, how manycycles are sufficient to determine the presence of a given targetpolynucleotide. As used herein, determining the presence of a target cancomprise identifying it, as well as optionally quantifying it. In someembodiments, the amplification products can be scored as positive ornegative as soon as a given number of cycles is complete. In someembodiments, the results may be transmitted electronically directly to adatabase and tabulated. Thus, in some embodiments, large numbers ofsamples can be processed and analyzed with less time and labor when suchan instrument is used. In some embodiments, different detector probesmay distinguish between different target polynucleotides. A non-limitingexample of such a probe is a 5′-nuclease fluorescent probe, such as aTaqMan® probe molecule, wherein a fluorescent molecule is attached to afluorescence-quenching molecule through an oligonucleotide link element.In some embodiments, the oligonucleotide link element of the 5′-nucleasefluorescent probe binds to a specific sequence of an identifying portionor its complement. In some embodiments, different 5′-nucleasefluorescent probes, each fluorescing at different wavelengths, candistinguish between different amplification products within the sameamplification reaction. For example, in some embodiments, one could usetwo different 5′-nuclease fluorescent probes that fluoresce at twodifferent wavelengths (WL_(A) and WL_(B)) and that are specific to twodifferent stem regions of two different extension reaction products (A′and B′, respectively). Amplification product A′ is formed if targetnucleic acid sequence A is in the sample, and amplification product B′is formed if target nucleic acid sequence B is in the sample. In someembodiments, amplification product A′ and/or B′ may form even if theappropriate target nucleic acid sequence is not in the sample, but suchoccurs to a measurably lesser extent than when the appropriate targetnucleic acid sequence is in the sample. After amplification, one candetermine which specific target nucleic acid sequences are present inthe sample based on the wavelength of signal detected and theirintensity. Thus, if an appropriate detectable signal value of onlywavelength WL_(A) is detected, one would know that the sample includestarget nucleic acid sequence A, but not target nucleic acid sequence B.If an appropriate detectable signal value of both wavelengths WL_(A) andWL_(B) are detected, one would know that the sample includes both targetnucleic acid sequence A and target nucleic acid sequence B. In someembodiments, detection can occur through any of a variety of mobilitydependent analytical techniques based on differential rates of migrationbetween different analyte species. Exemplary mobility-dependent analysistechniques include electrophoresis, chromatography, mass spectroscopy,sedimentation, e.g., gradient centrifugation, field-flow fractionation,multi-stage extraction techniques, and the like. In some embodiments,mobility probes can be hybridized to amplification products, and theidentity of the target polynucleotide determined via a mobilitydependent analysis technique of the eluted mobility probes, as describedfor example in Published P.C.T. Application WO04/46344 to Rosenblum etal., and WO01/92579 to Wenz et al. In some embodiments, detection can beachieved by various microarrays and related software such as the AppliedBiosystems Array System with the Applied Biosystems 1700Chemiluminescent Microarray Analyzer and other commercially availablearray systems available from Affymetrix, Agilent, Illumina, and AmershamBiosciences, among others (see also Gerry et al., J. Mol. Biol.292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; andStears et al., Nat. Med. 9:140-45, including supplements, 2003). It willalso be appreciated that detection can comprise reporter groups that areincorporated into the reaction products, either as part of labeledprimers or due to the incorporation of labeled dNTPs during anamplification, or attached to reaction products, for example but notlimited to, via hybridization tag complements comprising reporter groupsor via linker arms that are integral or attached to reaction products.Detection of unlabeled reaction products, for example using massspectrometry, is also within the scope of the current teachings.

Exemplary Embodiments

FIG. 1 depicts certain compositions according to some embodiments of thepresent teachings. Top, a miRNA molecule (1, dashed line) is depicted.Middle, a linker probe (2) is depicted, illustrating a 3′ targetspecific portion (3), a stem (4), and a loop (5). Bottom, a miRNAhybridized to a linker probe is depicted, illustrating the 3′ targetspecific portion of the linker probe (3) hybridized to the 3′ end regionof the miRNA (6).

As shown in FIG. 2, a target polynucleotide (9, dotted line) isillustrated to show the relationship with various components of thelinker probe (10), the detector probe (7), and the reverse primer (8),according to various non-limiting embodiments of the present teachings.For example as shown in FIG. 2A, in some embodiments the detector probe(7) can correspond with the 3′ end region of the target polynucleotidein the amplification product as well as a region upstream from the 3′end region of the target polynucleotide in the amplification product.(Here, the detector probe is depicted as rectangle (7) with an F and aQ, symbolizing a TaqMan probe with a florophore (F) and a quencher (Q)).Also shown in FIG. 2A, the loop can correspond to the reverse primer(8). In some embodiments as shown in FIG. 2B, the detector probe (7) cancorrespond with a region of the amplification product corresponding withthe 3′ end region of the target polynucleotide in the amplificationproduct, as well as a region upstream from the 3′ end region of thetarget polynucleotide in the amplification product, as well as thelinker probe stem in the amplification product. Also shown in FIG. 2B,the upstream region of the stem, as well as the loop, can correspond tothe reverse primer (8). In some embodiments as shown in FIG. 2C, thedetector probe can correspond to the amplification product in a mannersimilar to that shown in FIG. 2B, but the loop can correspond to thereverse primer (8). In some embodiments as shown in FIG. 2D, thedetector probe (7) can correspond with the linker probe stem in theamplification product. Also shown in FIG. 2D, the upstream region of thestem, as well as the loop can correspond to the reverse primer (8). Itwill be appreciated that various related strategies for implementing thedifferent functional regions of these compositions are possible in lightof the present teachings, and that such derivations are routine to onehaving ordinary skill in the art without undue experimentation.

FIG. 3 depicts the nucleotide relationship for the micro RNA MiR-16(boxed, 11) according to some embodiments of the present teachings.Shown here is the interrelationship of MiR-16 to a forward primer (12),a linker probe (13), a TaqMan detector probe (14), and a reverse primer(boxed, 15). The TaqMan probe comprises a 3′ minor groove binder (MGB),and a 5′ FAM florophore. It will be appreciated that in some embodimentsof the present teachings the detector probes, such as for example TaqManprobes, can hybridize to either strand of an amplification product. Forexample, in some embodiments the detector probe can hybridize to thestrand of the amplification product corresponding to the first strandsynthesized. In some embodiments, the detector probe can hybridize tothe strand of the amplification product corresponding to the secondstrand synthesized.

FIG. 4 depicts a single-plex assay design according to some embodimentsof the present teachings. Here, a miRNA molecule (16) and a linker probe(17) are hybridized together (18). The 3′ end of the linker probe of thetarget-linker probe composition is extended to form an extension product(19) that can be amplified in a PCR. The PCR can comprise a miRNAspecific forward primer (20) and a reverse primer (21). The detection ofa detector probe (22) during the amplification allows for quantitationof the miRNA.

FIG. 5 depicts an overview of a multiplex assay design according to someembodiments of the present teachings. Here, a multiplexed hybridizationand extension reaction is performed in a first reaction vessel (23).Thereafter, aliquots of the extension reaction products from the firstreaction vessel are transferred into a plurality of amplificationreactions (here, depicted as PCRs 1, 2, and 3) in a plurality of secondreaction vessels. Each PCR can comprise a distinct primer pair and adistinct detector probe. In some embodiments, a distinct primer pair butthe same detector probe can be present in each of a plurality of PCRs.

FIG. 6 depicts a multiplex assay design according to some embodiments ofthe present teachings. Here, three different miRNAs (24, 25, and 26) arequeried in a hybridization reaction comprising three different linkerprobes (27, 28, and 29). Following hybridization and extension to formextension products (30, 31, and 32), the extension products are dividedinto three separate amplification reactions. (Though not explicitlyshown, it will be appreciated that a number of copies of the moleculesdepicted by 30, 31, and 32 can be present, such that each of the threeamplification reactions can have copies of 30, 31, and 32.) PCR 1comprises a forward primer specific for miRNA 24 (33), PCR 2 comprises aforward primer specific for miRNA 25 (34), and PCR 3 comprises a forwardprimer specific for miRNA 26 (35). Each of the forward primers furthercomprise a non-complementary tail portion. PCR 1, PCR 2, and PCR 3 allcomprise the same universal reverse primer 36. Further, PCR 1 comprisesa distinct detector probe (37) that corresponds to the 3′ end region ofmiRNA 24 and the stem of linker probe 27, PCR 2 comprises a distinctdetector probe (38) that corresponds to the 3′ end region of miRNA 25and the stem of linker probe 28, and PCR 3 comprises a distinct detectorprobe (39) that corresponds to the 3′ region of miRNA 26 and the stem oflinker probe 29.

The present teachings also contemplate reactions comprisingconfigurations other than a linker probe. For example, in someembodiments, two hybridized molecules with a sticky end can be employed,wherein for example an overlapping 3′ sticky end hybridizes with the 3′end region of the target polynucleotide. Some descriptions of twomolecule configurations that can be employed in the present teachingscan be found in Chen et al., U.S. Provisional Application 60/517,470.Viewed in light of the present teachings herein, one of skill in the artwill appreciate that the approaches of Chen et al., can also be employedto result in extension reaction products that are longer that the targetpolynucleotide. These longer products can be detected with detectorprobes by, for example, taking advantage of the additional nucleotidesintroduced into the reaction products.

The present teachings also contemplate embodiments wherein the linkerprobe is ligated to the target polynucleotide, as described for examplein Chen et al., U.S. Provisional Application 60/575,661, and thecorresponding co-filed U.S. Provisional application co-filed herewith

Further, it will be appreciated that in some embodiments of the presentteachings, the two molecule configurations in Chen et al., U.S.Provisional Application 60/517,470 can be applied in embodimentscomprising the linker approaches discussed in Chen et al., U.S.Provisional Application 60/575,661.

Generally however, the loop structure of the present teachings willenhance the Tm of the target polynucleotide-linker probe duplex. Withoutbeing limited to any particular theory, this enhanced Tm could possiblybe due to base stacking effects. Also, the characteristics of the loopedlinker probe of the present teachings can minimize nonspecific primingduring the extension reaction, and/or a subsequent amplificationreaction such as PCR. Further, the looped linker probe of the presentteachings can better differentiate mature and precursor forms of miRNA,as illustrated infra in Example 6.

The present teachings also contemplate encoding and decoding reactionschemes, wherein a first encoding extension reaction is followed by asecond decoding amplification reaction, as described for example inLivak et al., U.S. Provisional Application 60/556,162, Chen et al., U.S.Provisional Application 60/556,157, Andersen et al., U.S. ProvisionalApplication 60/556,224, and Lao et al., U.S. Provisional Application60/556,163.

The present teachings also contemplate a variety of strategies tominimize the number of different molecules in multiplexed amplificationstrategies, as described for example in Whitcombe et al., U.S. Pat. No.6,270,967.

In certain embodiments, the present teachings also provide kits designedto expedite performing certain methods. In some embodiments, kits serveto expedite the performance of the methods of interest by assembling twoor more components used in carrying out the methods. In someembodiments, kits may contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits may include instructions for performing one or more methods of thepresent teachings. In certain embodiments, the kit components areoptimized to operate in conjunction with one another.

For example, the present teachings provide a kit comprising, a reversetranscriptase and a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′target-specific portion corresponds to a miRNA. In some embodiments, thekits can comprise a DNA polymerase. In some embodiments, the kits cancomprise a primer pair. In some embodiments, the kits can furthercomprise a forward primer specific for a miRNA, and, a universal reverseprimer, wherein the universal reverse primer comprises a nucleotide ofthe loop of the linker probe. In some embodiments, the kits can comprisea plurality of primer pairs, wherein each primer pair is in one reactionvessel of a plurality of reaction vessels. In some embodiments, the kitscan comprise a detector probe. In some embodiments, the detector probecomprises a nucleotide of the linker probe stem in the amplificationproduct or a nucleotide of the linker probe stem complement in theamplification product, and the detector probe further comprises anucleotide of the 3′ end region of the miRNA in the amplificationproduct or a nucleotide of the 3′ end region of the miRNA complement inthe amplification product.

The present teachings further contemplate kits comprising a means forhybridizing, a means for extending, a means for amplifying, a means fordetecting, or combinations thereof.

While the present teachings have been described in terms of theseexemplary embodiments, the skilled artisan will readily understand thatnumerous variations and modifications of these exemplary embodiments arepossible without undue experimentation. All such variations andmodifications are within the scope of the current teachings. Aspects ofthe present teachings may be further understood in light of thefollowing examples, which should not be construed as limiting the scopeof the teachings in any way.

EXAMPLE 1

A single-plex reaction was performed in replicate for a collection ofmouse miRNAs, and the effect of the presence or absence of ligase, aswell as the presence or absence of reverse transcriptase, determined.The results are shown in Table 1 as Ct values.

First, a 6 ul reaction was set up comprising: 1 ul Reverse TranscriptionEnzyme Mix (Applied Biosystems part number 4340444) (or 1 ul dH2O), 0.5ul T4 DNA Ligase (400 units/ul, NEB) (or 0.5 ul dH20), 0.25 ul 2M KCl,0.05 ul dNTPs (25 mM each), 0.25 ul T4 Kinase (10 units/ul, NEB), 1 ul10×T4 DNA ligase buffer (NEB), 0.25 ul Applied Biosystems RNaseInhibitor (10 units/ul), and 2.2 ul dH20 Next, 2 ul of the linker probe(0.25 uM) and RNA samples (2 ul of 0.25 ug/ul mouse lung total RNA(Ambion, product number 7818) were added. Next, the reaction was mixed,spun briefly, and placed on ice for 5 minutes.

The reaction was then incubated at 16 C for 30 minutes, 42 C for 30minutes, followed by 85 C for 5 minutes, and then held at 4 C. Thereactions were diluted 4 times by adding 30 ul of dH20 prior to the PCRamplification.

A 10 ul PCR amplification was then set up comprising: 2 ul of dilutedreverse transcription reaction product, 1.3 ul 10 uM miRNA specificForward Primer, 0.7 ul 10 uM Universal Reverse Primer, 0.2 ul TaqMandetector probe, 0.2 ul dNTPs (25 mM each), 0.6 ul dH20, 5 ul 2× TaqManmaster mix (Applied Biosystems, without UNG). The reaction was startedwith a 95 C step for 10 minutes. Then, 40 cycles were performed, eachcycle comprising 95 C for 15 seconds, and 60 C for 1 minute. Table 1indicates the results of this experiment.

TABLE 1 Reverse miRNA Replicate Ligase transcriptase Let-7a1 mir16 mir20mir21 mir26a mir30a mir224 average Yes Yes 16.8 16.0 19.1 16.8 15.0 21.327.3 18.9 Yes No 38.7 31.3 39.9 31.9 30.1 33.3 40.0 35.0 I No Yes 18.014.6 18.3 16.2 14.0 21.3 26.4 18.4 No No 40.0 36.6 40.0 40.0 33.8 39.240.0 38.5 Yes Yes 17.1 16.2 19.3 17.0 15.1 21.4 27.3 19.1 Yes No 38.931.2 37.6 32.1 30.4 33.4 39.4 34.7 II No Yes 18.4 14.8 18.7 16.6 14.321.5 26.7 18.7 No No 40.0 36.1 40.0 40.0 34.1 40.0 40.0 38.6 ReplicateYes Yes 16.9 16.1 19.2 16.9 15.0 21.4 27.3 19.0 Average Yes No 38.8 31.238.8 32.0 30.3 33.4 39.7 34.9 No Yes 18.2 14.7 18.5 16.4 14.1 21.4 26.618.6 No No 40.0 36.4 40.0 40.0 34.0 39.6 40.0 40.0

Sequences of corresponding forward primers, reverse primer, and TaqManprobes are shown in Table 2.

TABLE 2 miRNA ID SEQ ID NO: miRNA sequences miR-16 1uagcagcacguaaauauuggcg miR-20 2 uaaagugcuuauagugcaggua miR-21 3uagcuuaucagacugauguuga miR-22 4 aagcugccaguugaagaacugu miR-26a 5uucaaguaauccaggauaggcu miR-29 6 cuagcaccaucugaaaucgguu miR-30a 7cuuucagucggauguuugcagc miR-34 8 uggcagugucuuagcugguugu miR-200b 9cucuaauacugccugguaaugaug miR-323 10 gcacauuacacggucgaccucu miR-324-5 11cgcauccccuagggcauuggugu let-7a1 12 ugagguaguagguuguauaguu Linker probeSEQ ID NO: Linker probe sequences miR-16linR6 13GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCAC TGGATACGACCGCCAA miR20LinR6 14GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCAC TGGATACGACTACCTG miR-21linR6 15GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACT GGATACGACTCAACA miR-22linR6 16GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACT GGATACGACACAGTT miR-26alinR6 17GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACT GGATACGACAGCCTA miR-29linR6 18GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACAACCGA miR30LinR6 19GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACGCTGCA miR-34linR6 20GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACACAACC miR-200blinR6 21GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACCATCAT miR-323linR6 22GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACAGAGGT miR-324-5linR6 23GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACACACCA let7aLinR6 24GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG GATACGACAACTAT Forward primer IDSEQ ID NO: Forward primer sequences miR-16F55 25 CGCGCTAGCAGCACGTAAATmiR-20F56 26 GCCGCTAAAGTGCTTATAGTGC miR-21F56 27GCCCGCTAGCTTATCAGACTGATG miR-22F56 28 GCCTGAAGCTGCCAGTTGA miR-26aF54 29CCGGCGTTCAAGTAATCCAGGA miR-29F56 30 GCCGCTAGCACCATCTGAAA miR-30aF58 31GCCCCTTTCAGTCGGATGTTT miR-34F56 32 GCCCGTGGCAGTGTCTTAG miR-200bF56 33GCCCCTCTAATACTGCCTGG miR-323F58 34 GCCACGCACATTACACGGTC miR-324-5F56 35GCCACCATCCCCTAGGGC let-7a1F56 36 GCCGCTGAGGTAGTAGGTTGT TaqMan probe IDSEQ ID NO: TaqMan probe sequences miR-16_Tq8F67 37(6FAM) ATACGACCGCCAATAT (MGB) miR20_Tq8F68 38(6FAM) CTGGATACGACTACCTG (MGB) miR-21_Tq8F68 39(6FAM) CTGGATACGACTCAACA (MGB) miR-22_Tq8F68 40(6FAM) TGGATACGACACAGTTCT (MGB) miR-26a_Tq8F69 41(6FAM) TGGATACGACAGCCTATC (MGB) miR-29_Tq8F68 42(6FAM) TGGATACGACAACCGAT (MGB) miR30_Tq8F68 43(6FAM) CTGGATACGACGCTGC (MGB) miR-34_Tq8F68 44(6FAM) ATACGACACAACCAGC (MGB) miR-200b_Tq8F67 45(6FAM) ATACGACCATCATTACC (MGB) miR-323_Tq8F67 46(6FAM) CTGGATACGACAGAGGT (MGB) miR-324-5Tq8F68 47(6FAM) ATACGACACACCAATGC (MGB) let7a_Tq8F68 48(6FAM) TGGATACGACAACTATAC (MGB) Universal reverse primer ID SEQ ID NO:Reverse primer sequence miR-UP-R67.8 49 GTGCAGGGTCCGAGGT

EXAMPLE 2

A multiplex (12-plex) assay was performed and the results compared to acorresponding collection of single-plex reactions. Additionally, theeffect of the presence or absence of ligase, as well as the presence orabsence of reverse transcriptase, was determined. The experiments wereperformed essentially the same as in Example 1, and the concentration ofeach linker in the 12-plex reaction was 0.05 uM, thereby resulting in atotal linker probe concentration of 0.6 uM. Further, the diluted 12-plexreverse transcription product was split into 12 different PCRamplification reactions, wherein a miRNA forward primer and a universalreverse primer and a detector probe where in each amplificationreaction. The miRNA sequences, Forward primers, and TaqMan detectorprobes are included in Table 2. The results are shown in Table 3.

TABLE 3 Singleplex vs. Multiplex Assay With Or Without T4 DNA Ligase1-plex Ct 12-plex Ct Ligation + RT 1- vs. 12- miRNA Ligation + RT RTonly Ligation + RT RT only vs RT only plex let-7a1 17.8 16.3 17.6 17.01.0 −0.3 mir-16 16.0 15.1 16.1 15.3 0.9 −0.1 mir-20 19.3 18.7 19.8 19.50.4 −0.6 mir-21 17.0 15.8 17.1 16.3 1.0 −0.3 mir-22 21.6 20.4 21.4 20.71.0 −0.1 mir-26a 15.2 14.3 15.6 14.9 0.8 −0.4 mir-29 17.9 16.8 17.7 17.00.9 0.0 mir-30a 20.7 19.9 21.2 20.7 0.7 −0.7 mir-34 21.3 20.4 22.0 21.00.9 −0.6 mir-200b 19.9 19.2 21.1 20.2 0.8 −1.0 mir-323 32.5 31.2 33.632.3 1.3 −1.1 mir-324-5 24.7 23.1 25.0 24.4 1.1 −0.8 Average 20.3 19.320.7 19.9 0.9 −0.5

EXAMPLE 3

An experiment was performed to determine the effect of buffer conditionson reaction performance. In one set of experiments, a commerciallyavailable reverse transcription buffer from Applied Biosystems (partnumber 43400550) was employed in the hybridization and extensionreaction. In a corresponding set of experiments, a commerciallyavailable T4 DNA ligase buffer (NEB) was employed in the hybridizationand extension reaction. The experiments were performed as single-plexformat essentially as described for Example 1, and each miRNA was donein triplicate. The results are shown in Table 4, comparing RT buffer (ABpart #4340550) vs T4 DNA ligase buffer.

TABLE 4 RT Buffer T4 DNA Ligase Buffer RT vs I II III Mean I II III MeanT4 Buffer let-7a1 22.7 22.8 22.8 22.8 20.8 20.7 20.6 20.7 2.1 mir-1618.4 18.5 18.6 18.5 17.7 17.8 17.9 17.8 0.7 mir-20 23.6 23.7 23.8 23.723.1 23.1 23.0 23.1 0.6 mir-21 20.4 20.4 20.5 20.4 19.4 19.3 19.2 19.31.1 mir-22 24.0 23.9 24.1 24.0 22.7 22.7 22.7 22.7 1.3 mir-26a 19.8 19.920.1 19.9 18.9 19.0 19.0 18.9 1.0 mir-29 21.3 21.3 21.4 21.3 20.5 20.620.5 20.5 0.8 mir-30a 24.4 24.4 24.4 24.4 23.6 23.4 23.6 23.5 0.9 mir-3424.9 24.8 25.1 25.0 23.0 23.1 23.2 23.1 1.9 mir-200b 25.8 25.8 25.9 25.924.6 24.6 24.8 24.7 1.2 mir-323 34.6 34.5 34.8 34.6 34.7 34.2 34.5 34.50.2 mir-324-5 26.0 26.0 26.1 26.0 25.4 25.7 25.6 25.6 0.5 Average 23.823.8 24.0 23.9 22.9 22.8 22.9 22.9 1.0

EXAMPLE 4

An experiment was performed to examine the effect of ligase and kinasein a real-time miRNA amplification reaction. Here, twelve single-plexreactions were performed in duplicate, essentially as described inExample 1. Results are shown in Table 5.

TABLE 5 Ligase & Kinase No Ligase/No Kinase I II Mean I II Mean let-7a117.7 17.9 17.8 16.2 16.4 16.3 mir-16 15.9 16.2 16.0 15.0 15.2 15.1mir-20 19.1 19.6 19.3 18.6 18.9 18.7 mir-21 16.9 17.2 17.0 15.7 15.915.8 mir-22 21.4 21.7 21.6 20.3 20.5 20.4 mir-26a 15.0 15.4 15.2 14.314.4 14.3 mir-29 17.9 18.0 17.9 16.7 16.8 16.8 mir-30a 20.6 20.8 20.719.8 20.0 19.9 mir-34 21.1 21.5 21.3 20.4 20.5 20.4 mir-200b 19.8 20.019.9 19.2 19.3 19.2 mir-323 32.3 32.6 32.5 31.1 31.2 31.2 mir-324-5 24.624.8 24.7 23.0 23.3 23.1 Average 20.2 20.5 20.3 19.2 19.4 19.3

EXAMPLE 5

An experiment was performed to determine the effect of sample materialon Ct values in a real-time miRNA amplification reaction. Here, cells,GuHCl lysate, Tris lysate, and Purified RNA were compared. The cellswere NIH3T3 cells. The Purified RNA was collected using the commerciallyavailable mirVana mRNA isolation kit for Ambion (catalog number 1560). ATris lysate, and a Guanidine lysate (GuHCl) (commercially available fromApplied Biosystems), were prepared as follows:

For the Tris lysate, a 1× lysis buffer comprised 10 mM Tris-HCl, pH 8.0,0.02% Sodium Azide, and 0.03% Tween-20. Trypsinized cells were pelletedby centrifugation at 1500 rpm for 5 minutes. The growth media wasremoved by aspiration, being careful that the cell pellet was notdisturbed. PBS was added to bring the cells to 2×10³ cells/ul. Next 10ul of cell suspension was mixed with 10 ul of a 2× lysis buffer and spunbriefly. The tubes were then immediately incubated for 5 minutes at 95C, and then immediately placed in a chilled block on ice for 2 minutes.The tubes were then mixed well and spun briefly at full speed before use(or optionally, stored at −20 C).

For the GuHCl lysate, a 1× lysis buffer comprised 2.5M GuHCl, 150 mM MESpH 6.0, 200 mM NaCl, 0.75% Tween-20. Trypsinized cells were pelleted bycentrifugation at 1500 rpm for 5 minutes. The growth media was removedby aspiration, being careful that the cell pellet was not disturbed. Thecell pellet was then re-suspended in 1×PBS, Ca++ and Mg++free to bringcells to 2×10⁴ cells/uL. Then, 1 volume of 2× lysis buffer was added. Toensure complete nucleic acid release, this was followed by pipetting upand down ten times, followed by a brief spin. Results are shown in Table6.

Similar results were obtained for a variety of cell lines, including.NIH/3T3, OP9, A549, and HepG2 cells.

TABLE 6 Ct miRNA ID Cells GuHCl lysate Tris lysate Purified RNA let-7a124.9 31.3 28.2 31.5 mir-16 22.3 25.2 22.3 24.9 mir-20 22.7 26.0 24.126.1 mir-21 21.3 24.2 22.0 24.7 mir-22 30.3 28.6 27.2 28.8 mir-26a 25.631.0 27.9 31.4 mir-29 27.2 27.9 26.5 27.4 mir-30a 26.1 32.2 28.9 30.7mir-34 26.8 30.3 26.4 27.4 mir-200b 40.0 40.0 40.0 40.0 mir-323 30.134.7 31.1 31.8 mir-324-5 28.6 29.7 28.3 29.3 Average 27.2 30.1 27.8 29.5

EXAMPLE 6

An experiment was performed to demonstrate the ability of the reactionto selectively quantity mature miRNA in the presence of precursor miRNA.Here, let-7a miRNA and mir-26b miRNA were queried in both mature form aswell as in their precursor form. Experiments were performed essentiallyas described for Example 1 in the no ligase condition, done intriplicate, with varying amounts of target material as indicated.Results are shown in Table 7. The sequences examined were as follows:

Mature let-7a, Seq ID NO: 50 UGAGGUAGUAGGUUGUAUAGUUPrecursor let-7a, SEQ ID NO: 51 (Note that theunderlined sequences corresponds to the Mature let-7a.)GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGAUAACUAUACAAUCUACUGUCUUUCCU Mature mir-26b, SEQ ID NO: 52UUCAAGUAAUUCAGGAUAGGU Precursor mir-26b of SEQ ID NO: 53 (Note that theunderlined sequences corresponds to the Mature mir-26b.)CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGCUCGGGGACCGG

TABLE 7 Mouse Synthetic Synthetic Assay lung miRNA precursor specificfor (CT) Target RNA (ng) (fM) (fM) miRNA Precursor Let-7a 0 0 0 40.0 ±0.0 40.0 ± 0.0 (let-7a3) 0 10 0 24.2 ± 0.3 40.0 ± 0.0 0 100 0 21.0 ± 0.240.0 ± 0.0 0 0 10 35.0 ± 1.0 25.0 ± 0.1 0 0 100 31.0 ± 0.1 21.5 ± 0.1 100 0 19.1 ± 0.4 40.0 ± 0.0 Mir-26b 0 0 0 40.0 ± 0.0 40.0 ± 0.0 0 10 023.1 ± 0.1 40.0 ± 0.0 0 100 0 19.7 ± 0.1 40.0 ± 0.0 0 0 10 32.9 ± 0.425.7 ± 0.0 0 0 100 28.9 ± 0.2 22.3 ± 0.0 10 0 0 20.5 ± 0.1 28.0 ± 0.2

EXAMPLE 7

An experiment was performed on synthetic let-7a miRNA to assess thenumber of 3′ nucleotides in the 3′ target specific portion of the linkerprobe that correspond with the 3′ end region of the miRNA. Theexperiment was performed as essentially as described supra for Example 1for the no ligase condition, and results are shown in Table 8 as meansand standard deviations of Ct values.

TABLE 8 miRNA assay components: let-7a miRNA synthetic target: let-7aNo. 3′ ssDNA linker probe target C_(T) values & statistics specificportion bases I II III Average SD 7 29.4 29.1 29.3 29.3 0.1 6 30.1 29.930.2 30.1 0.2 5 33.9 33.2 33.8 33.6 0.4 4 40.0 39.2 40.0 39.7 0.4In some embodiments, 3′ target specific portions of linker probespreferably comprise 5 nucleotides that correspond to the 3′ end regionof miRNAs. For example, miR-26a and miR-26b differ by only 2 bases, oneof which is the 3′ end nucleotide of miR-26a. Linker probes comprising 5nucleotides at their 3′ target specific portions can be employed toselectively detect miR-26a versus miR-26b.

Additional strategies for using the linker probes of the presentteachings in the context of single step assays, as well as in thecontext of short primer compositions, can be found in filed U.S.Provisional Application “Compositions, Methods, and Kits for Identifyingand Quantitating Small RNA Molecules” by Lao and Straus, as well as inElfaitouri et al., J. Clin. Virol. 2004, 30(2): 150-156.

The present teachings further contemplate linker probe compositionscomprising 3′ target specific portions corresponding to any micro RNAsequence, including but without limitation, those sequences shown inTable 9, including C. elegans (cel), mouse (mmu), human (hsa),drosophila (dme), rat (mo), and rice (osa).

TABLE 9 cel-let-7 SEQ ID NO: mmu-let-7g SEQ ID NO:ugagguaguagguuguauaguu 54 ugagguaguaguuuguacagu 348 cel-lin-4 mmu-let-7iucccugagaccucaaguguga 55 ugagguaguaguuugugcu 349 cel-miR-1 mmu-miR-1uggaauguaaagaaguaugua 56 uggaauguaaagaaguaugua 350 cel-miR-2 mmu-miR-15buaucacagccagcuuugaugugc 57 uagcagcacaucaugguuuaca 351 cel-miR-34mmu-miR-23b aggcagugugguuagcugguug 58 aucacauugccagggauuaccac 352cel-miR-35 mmu-miR-27b ucaccggguggaaacuagcagu 59 uucacaguggcuaaguucug353 cel-miR-36 mmu-miR-29b ucaccgggugaaaauucgcaug 60uagcaccauuugaaaucagugu 354 cel-miR-37 mmu-miR-30a*ucaccgggugaacacuugcagu 61 uguaaacauccucgacuggaagc 355 cel-miR-38mmu-miR-30a ucaccgggagaaaaacuggagu 62 cuuucagucggauguuugcagc 356cel-miR-39 mmu-miR-30b ucaccggguguaaaucagcuug 63 uguaaacauccuacacucagc357 cel-miR-40 mmu-miR-99a ucaccggguguacaucagcuaa 64acccguagauccgaucuugu 358 cel-miR-41 mmu-miR-99b ucaccgggugaaaaaucaccua65 cacccguagaaccgaccuugcg 359 cel-miR-42 mmu-miR-101caccggguuaacaucuacag 66 uacaguacugugauaacuga 360 cel-miR-43 mmu-miR-124auaucacaguuuacuugcugucgc 67 uuaaggcacgcggugaaugcca 361 cel-miR-44mmu-miR-125a ugacuagagacacauucagcu 68 ucccugagacccuuuaaccugug 362cel-miR-45 mmu-miR-125b ugacuagagacacauucagcu 69 ucccugagacccuaacuuguga363 cel-miR-46 mmu-miR-126* ugucauggagucgcucucuuca 70cauuauuacuuuugguacgcg 364 cel-miR-47 mmu-miR-126 ugucauggaggcgcucucuuca71 ucguaccgugaguaauaaugc 365 cel-miR-48 mmu-miR-127ugagguaggcucaguagaugcga 72 ucggauccgucugagcuuggcu 366 cel-miR-49mmu-miR-128a aagcaccacgagaagcugcaga 73 ucacagugaaccggucucuuuu 367cel-miR-50 mmu-miR-130a ugauaugucugguauucuuggguu 74 cagugcaauguuaaaagggc368 cel-miR-51 mmu-miR-9 uacccguagcuccuauccauguu 75ucuuugguuaucuagcuguauga 369 cel-miR-52 mmu-miR-9*cacccguacauauguuuccgugcu 76 uaaagcuagauaaccgaaagu 370 cel-miR-53mmu-miR-132 cacccguacauuuguuuccgugcu 77 uaacagucuacagccauggucg 371cel-miR-54 mmu-miR-133a uacccguaaucuucauaauccgag 78uugguccccuucaaccagcugu 372 cel-miR-55 mmu-miR-134uacccguauaaguuucugcugag 79 ugugacugguugaccagaggg 373 cel-miR-56*mmu-miR-135a uggcggauccauuuuggguugua 80 uauggcuuuuuauuccuauguga 374cel-miR-56 mmu-miR-136 uacccguaauguuuccgcugag 81 acuccauuuguuuugaugaugga375 cel-miR-57 mmu-miR-137 uacccuguagaucgagcugugugu 82uauugcuuaagaauacgcguag 376 cel-miR-58 mmu-miR-138 ugagaucguucaguacggcaau83 agcugguguugugaauc 377 cel-miR-59 mmu-miR-140 ucgaaucguuuaucaggaugaug84 agugguuuuacccuaugguag 378 cel-miR-60 mmu-miR-141uauuaugcacauuuucuaguuca 85 aacacugucugguaaagaugg 379 cel-miR-61mmu-miR-142-5p ugacuagaaccguuacucaucuc 86 cauaaaguagaaagcacuac 380cel-miR-62 mmu-miR-142-3p ugauauguaaucuagcuuacag 87uguaguguuuccuacuuuaugg 381 cel-miR-63 mmu-miR-144uaugacacugaagcgaguuggaaa 88 uacaguauagaugauguacuag 382 cel-miR-64mmu-miR-145 uaugacacugaagcguuaccgaa 89 guccaguuuucccaggaaucccuu 383cel-miR-65 mmu-miR-146 uaugacacugaagcguaaccgaa 90 ugagaacugaauuccauggguu384 cel-miR-66 mmu-miR-149 caugacacugauuagggauguga 91ucuggcuccgugucuucacucc 385 cel-miR-67 mmu-miR-150ucacaaccuccuagaaagaguaga 92 ucucccaacccuuguaccagug 386 cel-miR-68mmu-miR-151 ucgaagacucaaaaguguaga 93 cuagacugaggcuccuugagg 387cel-miR-69 mmu-miR-152 ucgaaaauuaaaaaguguaga 94 ucagugcaugacagaacuugg388 cel-miR-70 mmu-miR-153 uaauacgucguugguguuuccau 95uugcauagucacaaaaguga 389 cel-miR-71 mmu-miR-154 ugaaagacauggguaguga 96uagguuauccguguugccuucg 390 cel-miR-72 mmu-miR-155 aggcaagauguuggcauagc97 uuaaugcuaauugugauagggg 391 cel-miR-73 mmu-miR-10buggcaagauguaggcaguucagu 98 cccuguagaaccgaauuugugu 392 cel-miR-74mmu-miR-129 uggcaagaaauggcagucuaca 99 cuuuuugcggucugggcuugcu 393cel-miR-75 mmu-miR-181a uuaaagcuaccaaccggcuuca 100aacauucaacgcugucggugagu 394 cel-miR-76 mmu-miR-182uucguuguugaugaagccuuga 101 uuuggcaaugguagaacucaca 395 cel-miR-77mmu-miR-183 uucaucaggccauagcugucca 102 uauggcacugguagaauucacug 396cel-miR-78 mmu-miR-184 uggaggccugguuguuugugc 103 uggacggagaacugauaagggu397 cel-miR-79 mmu-miR-185 auaaagcuagguuaccaaagcu 104 uggagagaaaggcaguuc398 cel-miR-227 mmu-miR-186 agcuuucgacaugauucugaac 105caaagaauucuccuuuugggcuu 399 cel-miR-80 mmu-miR-187ugagaucauuaguugaaagccga 106 ucgugucuuguguugcagccgg 400 cel-miR-81mmu-miR-188 ugagaucaucgugaaagcuagu 107 caucccuugcaugguggagggu 401cel-miR-82 mmu-miR-189 ugagaucaucgugaaagccagu 108gugccuacugagcugauaucagu 402 cel-miR-83 mmu-miR-24 uagcaccauauaaauucaguaa109 uggcucaguucagcaggaacag 403 cel-miR-84 mmu-miR-190ugagguaguauguaauauugua 110 ugauauguuugauauauuaggu 404 cel-miR-85mmu-miR-191 uacaaaguauuugaaaagucgugc 111 caacggaaucccaaaagcagcu 405cel-miR-86 mmu-miR-193 uaagugaaugcuuugccacaguc 112 aacuggccuacaaagucccag406 cel-miR-87 mmu-miR-194 gugagcaaaguuucaggugu 113uguaacagcaacuccaugugga 407 cel-miR-90 mmu-miR-195 ugauauguuguuugaaugcccc114 uagcagcacagaaauauuggc 408 cel-miR-124 mmu-miR-199auaaggcacgcggugaaugcca 115 cccaguguucagacuaccuguuc 409 cel-miR-228mmu-miR-199a* aauggcacugcaugaauucacgg 116 uacaguagucugcacauugguu 410cel-miR-229 mmu-miR-200b aaugacacugguuaucuuuuccaucgu 117uaauacugccugguaaugaugac 411 cel-miR-230 mmu-miR-201guauuaguugugcgaccaggaga 118 uacucaguaaggcauuguucu 412 cel-miR-231mmu-miR-202 uaagcucgugaucaacaggcagaa 119 agagguauagcgcaugggaaga 413cel-miR-232 mmu-miR-203 uaaaugcaucuuaacugcgguga 120ugaaauguuuaggaccacuag 414 cel-miR-233 mmu-miR-204uugagcaaugcgcaugugcggga 121 uucccuuugucauccuaugccug 415 cel-miR-234mmu-miR-205 uuauugcucgagaauacccuu 122 uccuucauuccaccggagucug 416cel-miR-235 mmu-miR-206 uauugcacucuccccggccuga 123uggaauguaaggaagugugugg 417 cel-miR-236 mmu-miR-207uaauacugucagguaaugacgcu 124 gcuucuccuggcucuccucccuc 418 cel-miR-237mmu-miR-122a ucccugagaauucucgaacagcuu 125 uggagugugacaaugguguuugu 419cel-miR-238 mmu-miR-143 uuuguacuccgaugccauucaga 126ugagaugaagcacuguagcuca 420 cel-miR-239a mmu-miR-30euuuguacuacacauagguacugg 127 uguaaacauccuugacugga 421 cel-miR-239bmmu-miR-290 uuguacuacacaaaaguacug 128 cucaaacuaugggggcacuuuuu 422cel-miR-240 mmu-miR-291-5p uacuggcccccaaaucuucgcu 129caucaaaguggaggcccucucu 423 cel-miR-241 mmu-miR-291-3pugagguaggugcgagaaauga 130 aaagugcuuccacuuugugugcc 424 cel-miR-242mmu-miR-292-5p uugcguaggccuuugcuucga 131 acucaaacugggggcucuuuug 425cel-miR-243 mmu-miR-292-3p cgguacgaucgcggcgggauauc 132aagugccgccagguuuugagugu 426 cel-miR-244 mmu-miR-293ucuuugguuguacaaagugguaug 133 agugccgcagaguuuguagugu 427 cel-miR-245mmu-miR-294 auugguccccuccaaguagcuc 134 aaagugcuucccuuuugugugu 428cel-miR-246 mmu-miR-295 uuacauguuucggguaggagcu 135aaagugcuacuacuuuugagucu 429 cel-miR-247 mmu-miR-296ugacuagagccuauucucuucuu 136 agggcccccccucaauccugu 430 cel-miR-248mmu-miR-297 uacacgugcacggauaacgcuca 137 auguaugugugcaugugcaug 431cel-miR-249 mmu-miR-298 ucacaggacuuuugagcguugc 138ggcagaggagggcuguucuucc 432 cel-miR-250 mmu-miR-299ucacagucaacuguuggcaugg 139 ugguuuaccgucccacauacau 433 cel-miR-251mmu-miR-300 uuaaguaguggugccgcucuuauu 140 uaugcaagggcaagcucucuuc 434cel-miR-252 mmu-miR-301 uaaguaguagugccgcagguaac 141cagugcaauaguauugucaaagc 435 cel-miR-253 mmu-miR-302cacaccucacuaacacugacc 142 uaagugcuuccauguuuugguga 436 cel-miR-254mmu-miR-34c ugcaaaucuuucgcgacuguagg 143 aggcaguguaguuagcugauugc 437cel-miR-256 mmu-miR-34b uggaaugcauagaagacugua 144uaggcaguguaauuagcugauug 438 cel-miR-257 mmu-let-7dgaguaucaggaguacccaguga 145 agagguaguagguugcauagu 439 cel-miR-258mmu-let-7d* gguuuugagaggaauccuuuu 146 cuauacgaccugcugccuuucu 440cel-miR-259 mmu-miR-106a aaaucucauccuaaucuggua 147caaagugcuaacagugcaggua 441 cel-miR-260 mmu-miR-106b gugaugucgaacucuuguag148 uaaagugcugacagugcagau 442 cel-miR-261 mmu-miR-130buagcuuuuuaguuuucacg 149 cagugcaaugaugaaagggcau 443 cel-miR-262mmu-miR-19b guuucucgauguuuucugau 150 ugugcaaauccaugcaaaacuga 444cel-miR-264 mmu-miR-30c ggcgggugguuguuguuaug 151 uguaaacauccuacacucucagc445 cel-miR-265 mmu-miR-30d ugagggaggaagggugguau 152uguaaacauccccgacuggaag 446 cel-miR-266 mmu-miR-148a aggcaagacuuuggcaaagc153 ucagugcacuacagaacuuugu 447 cel-miR-267 mmu-miR-192cccgugaagugucugcugca 154 cugaccuaugaauugaca 448 cel-miR-268 mmu-miR-196ggcaagaauuagaagcaguuuggu 155 uagguaguuucauguuguugg 449 cel-miR-269mmu-miR-200a ggcaagacucuggcaaaacu 156 uaacacugucugguaacgaugu 450cel-miR-270 mmu-miR-208 ggcaugauguagcaguggag 157 auaagacgagcaaaaagcuugu451 cel-miR-271 mmu-let-7a ucgccgggugggaaagcauu 158ugagguaguagguuguauaguu 452 cel-miR-272 mmu-let-7b uguaggcauggguguuug 159ugagguaguagguugugugguu 453 cel-miR-273 mmu-let-7c ugcccguacugugucggcug160 ugagguaguagguuguaugguu 454 cel-miR-353 mmu-let-7ecaauugccauguguugguauu 161 ugagguaggagguuguauagu 455 cel-miR-354mmu-let-7f accuuguuuguugcugcuccu 162 ugagguaguagauuguauaguu 456cel-miR-355 mmu-miR-15a uuuguuuuagccugagcuaug 163 uagcagcacauaaugguuugug457 cel-miR-356 mmu-miR-16 uugagcaacgcgaacaaauca 164uagcagcacguaaauauuggcg 458 cel-miR-357 mmu-miR-18 uaaaugccagucguugcagga165 uaaggugcaucuagugcagaua 459 cel-miR-358 mmu-miR-20caauugguaucccugucaagg 166 uaaagugcuuauagugcagguag 460 cel-miR-359mmu-miR-21 ucacuggucuuucucugacga 167 uagcuuaucagacugauguuga 461cel-miR-360 mmu-miR-22 ugaccguaaucccguucacaa 168 aagcugccaguugaagaacugu462 cel-lsy-6 mmu-miR-23a uuuuguaugagacgcauuucg 169aucacauugccagggauuucc 463 cel-miR-392 mmu-miR-26a uaucaucgaucacgugugauga170 uucaaguaauccaggauaggcu 464 mmu-miR-26b uucaaguaauucaggauagguu 465hsa-let-7a mmu-miR-29a ugagguaguagguuguauaguu 171 cuagcaccaucugaaaucgguu466 hsa-let-7b mmu-miR-29c ugagguaguagguugugugguu 172uagcaccauuugaaaucgguua 467 hsa-let-7c mmu-miR-27a ugagguaguagguuguaugguu173 uucacaguggcuaaguuccgc 468 hsa-let-7d mmu-miR-31agagguaguagguugcauagu 174 aggcaagaugcuggcauagcug 469 hsa-let-7emmu-miR-92 ugagguaggagguuguauagu 175 uauugcacuugucccggccug 470hsa-let-7f mmu-miR-93 ugagguaguagauuguauaguu 176 caaagugcuguucgugcagguag471 hsa-miR-15a mmu-miR-96 uagcagcacauaaugguuugug 177uuuggcacuagcacauuuuugcu 472 hsa-miR-16 mmu-miR-34auagcagcacguaaauauuggcg 178 uggcagugucuuagcugguuguu 473 hsa-miR-17-5pmmu-miR-98 caaagugcuuacagugcagguagu 179 ugagguaguaaguuguauuguu 474hsa-miR-17-3p mmu-miR-103 acugcagugaaggcacuugu 180agcagcauuguacagggcuauga 475 hsa-miR-18 mmu-miR-323uaaggugcaucuagugcagaua 181 gcacauuacacggucgaccucu 476 hsa-miR-19ammu-miR-324-5p ugugcaaaucuaugcaaaacuga 182 cgcauccccuagggcauuggugu 477hsa-miR-19b mmu-miR-324-3p ugugcaaauccaugcaaaacuga 183ccacugccccaggugcugcugg 478 hsa-miR-20 mmu-miR-325 uaaagugcuuauagugcaggua184 ccuaguaggugcucaguaagugu 479 hsa-miR-21 mmu-miR-326uagcuuaucagacugauguuga 185 ccucugggcccuuccuccagu 480 hsa-miR-22mmu-miR-328 aagcugccaguugaagaacugu 186 cuggcccucucugcccuuccgu 481hsa-miR-23a mmu-miR-329 aucacauugccagggauuucc 187 aacacacccagcuaaccuuuuu482 hsa-miR-189 mmu-miR-330 gugccuacugagcugauaucagu 188gcaaagcacagggccugcagaga 483 hsa-miR-24 mmu-miR-331uggcucaguucagcaggaacag 189 gccccugggccuauccuagaa 484 hsa-miR-25mmu-miR-337 cauugcacuugucucggucuga 190 uucagcuccuauaugaugccuuu 485hsa-miR-26a mmu-miR-338 uucaaguaauccaggauaggcu 191uccagcaucagugauuuuguuga 486 hsa-miR-26b mmu-miR-339uucaaguaauucaggauaggu 192 ucccuguccuccaggagcuca 487 hsa-miR-27ammu-miR-340 uucacaguggcuaaguuccgcc 193 uccgucucaguuacuuuauagcc 488hsa-miR-28 mmu-miR-341 aaggagcucacagucuauugag 194 ucgaucggucggucggucagu489 hsa-miR-29a mmu-miR-342 cuagcaccaucugaaaucgguu 195ucucacacagaaaucgcacccguc 490 hsa-miR-30a* mmu-miR-344uguaaacauccucgacuggaagc 196 ugaucuagccaaagccugacugu 491 hsa-miR-30ammu-miR-345 cuuucagucggauguuugcagc 197 ugcugaccccuaguccagugc 492hsa-miR-31 mmu-miR-346 ggcaagaugcuggcauagcug 198 ugucugcccgagugccugccucu493 hsa-miR-32 mmu-miR-350 uauugcacauuacuaaguugc 199uucacaaagcccauacacuuucac 494 hsa-miR-33 mmu-miR-135b gugcauuguaguugcauug200 uauggcuuuucauuccuaugug 495 hsa-miR-92 mmu-miR-101buauugcacuugucccggccugu 201 uacaguacugugauagcugaag 496 hsa-miR-93mmu-miR-107 aaagugcuguucgugcagguag 202 agcagcauuguacagggcuauca 497hsa-miR-95 mmu-miR-10a uucaacggguauuuauugagca 203uacccuguagauccgaauuugug 498 hsa-miR-96 mmu-miR-17-5puuuggcacuagcacauuuuugc 204 caaagugcuuacagugcagguagu 499 hsa-miR-98mmu-miR-17-3p ugagguaguaaguuguauuguu 205 acugcagugagggcacuugu 500hsa-miR-99a mmu-miR-19a aacccguagauccgaucuugug 206ugugcaaaucuaugcaaaacuga 501 hsa-miR-100 mmu-miR-25aacccguagauccgaacuugug 207 cauugcacuugucucggucuga 502 hsa-miR-101mmu-miR-28 uacaguacugugauaacugaag 208 aaggagcucacagucuauugag 503hsa-miR-29b mmu-miR-32 uagcaccauuugaaaucagu 209 uauugcacauuacuaaguugc504 hsa-miR-103 mmu-miR-100 agcagcauuguacagggcuauga 210aacccguagauccgaacuugug 505 hsa-miR-105 mmu-miR-139 ucaaaugcucagacuccugu211 ucuacagugcacgugucu 506 hsa-miR-106a mmu-miR-200caaaagugcuuacagugcagguagc 212 aauacugccggguaaugaugga 507 hsa-miR-107mmu-miR-210 agcagcauuguacagggcuauca 213 cugugcgugugacagcggcug 508hsa-miR-192 mmu-miR-212 cugaccuaugaauugacagcc 214 uaacagucuccagucacggcc509 hsa-miR-196 mmu-miR-213 uagguaguuucauguuguugg 215accaucgaccguugauuguacc 510 hsa-miR-197 mmu-miR-214uucaccaccuucuccacccagc 216 acagcaggcacagacaggcag 511 hsa-miR-198mmu-miR-216 gguccagaggggagauagg 217 uaaucucagcuggcaacugug 512hsa-miR-199a mmu-miR-218 cccaguguucagacuaccuguuc 218uugugcuugaucuaaccaugu 513 hsa-miR-199a* mmu-miR-219uacaguagucugcacauugguu 219 ugauuguccaaacgcaauucu 514 hsa-miR-208mmu-miR-223 auaagacgagcaaaaagcuugu 220 ugucaguuugucaaauacccc 515hsa-miR-148a mmu-miR-320 ucagugcacuacagaacuuugu 221aaaagcuggguugagagggcgaa 516 hsa-miR-30c mmu-miR-321uguaaacauccuacacucucagc 222 uaagccagggauuguggguuc 517 hsa-miR-30dmmu-miR-33 uguaaacauccccgacuggaag 223 gugcauuguaguugcauug 518hsa-miR-139 mmu-miR-211 ucuacagugcacgugucu 224 uucccuuugucauccuuugccu519 hsa-miR-147 mmu-miR-221 guguguggaaaugcuucugc 225agcuacauugucugcuggguuu 520 hsa-miR-7 mmu-miR-222 uggaagacuagugauuuuguu226 agcuacaucuggcuacugggucu 521 hsa-miR-10a mmu-miR-224uacccuguagauccgaauuugug 227 uaagucacuagugguuccguuua 522 hsa-miR-10bmmu-miR-199b uacccuguagaaccgaauuugu 228 cccaguguuuagacuaccuguuc 523hsa-miR-34a mmu-miR-181b uggcagugucuuagcugguugu 229aacauucauugcugucgguggguu 524 hsa-miR-181a mmu-miR-181caacauucaacgcugucggugagu 230 aacauucaaccugucggugagu 525 hsa-miR-181bmmu-miR-128b aacauucauugcugucgguggguu 231 ucacagugaaccggucucuuuc 526hsa-miR-181c mmu-miR-7 aacauucaaccugucggugagu 232 uggaagacuagugauuuuguu527 hsa-miR-182 mmu-miR-7b uuuggcaaugguagaacucaca 233uggaagacuugugauuuuguu 528 hsa-miR-182* mmu-miR-217 ugguucuagacuugccaacua234 uacugcaucaggaacugacuggau 529 hsa-miR-183 mmu-miR-133buauggcacugguagaauucacug 235 uugguccccuucaaccagcua 530 hsa-miR-187mmu-miR-215 ucgugucuuguguugcagccg 236 augaccuaugauuugacagac 531hsa-miR-199b cccaguguuuagacuaucuguuc 237 hsa-miR-203 dme-miR-1gugaaauguuuaggaccacuag 238 uggaauguaaagaaguauggag 532 hsa-miR-204dme-miR-2a uucccuuugucauccuaugccu 239 uaucacagccagcuuugaugagc 533hsa-miR-205 dme-miR-2b uccuucauuccaccggagucug 240uaucacagccagcuuugaggagc 534 hsa-miR-210 dme-miR-3 cugugcgugugacagcggcug241 ucacugggcaaagugugucuca 535 hsa-miR-211 dme-miR-4uucccuuugucauccuucgccu 242 auaaagcuagacaaccauuga 536 hsa-miR-212dme-miR-5 uaacagucuccagucacggcc 243 aaaggaacgaucguugugauaug 537hsa-miR-213 dme-miR-6 accaucgaccguugauuguacc 244 uaucacaguggcuguucuuuuu538 hsa-miR-214 dme-miR-7 acagcaggcacagacaggcag 245uggaagacuagugauuuuguugu 539 hsa-miR-215 dme-miR-8 augaccuaugaauugacagac246 uaauacugucagguaaagauguc 540 hsa-miR-216 dme-miR-9auaaucucagcuggcaacugug 247 ucuuugguuaucuagcuguauga 541 hsa-miR-217dme-miR-10 uacugcaucaggaacugauuggau 248 acccuguagauccgaauuugu 542hsa-miR-218 dme-miR-11 uugugcuugaucuaaccaugu 249 caucacagucugaguucuugc543 hsa-miR-219 dme-miR-12 ugauuguccaaacgcaauucu 250ugaguauuacaucagguacuggu 544 hsa-miR-220 dme-miR-13accacaccguaucugacacuuu 251 uaucacagccauuuugaugagu 545 hsa-miR-221dme-miR-13b agcuacauugucugcuggguuuc 252 uaucacagccauuuugacgagu 546hsa-miR-222 dme-miR-14 agcuacaucuggcuacugggucuc 253ucagucuuuuucucucuccua 547 hsa-miR-223 dme-miR-263a ugucaguuugucaaauacccc254 guuaauggcacuggaagaauucac 548 hsa-miR-224 dme-miR-184*caagucacuagugguuccguuua 255 ccuuaucauucucucgccccg 549 hsa-miR-200bdme-miR-184 cucuaauacugccugguaaugaug 256 uggacggagaacugauaagggc 550hsa-let-7g dme-miR-274 ugagguaguaguuuguacagu 257uuuugugaccgacacuaacggguaau 551 hsa-let-7i dme-miR-275ugagguaguaguuugugcu 258 ucagguaccugaaguagcgcgcg 552 hsa-miR-1dme-miR-92a uggaauguaaagaaguaugua 259 cauugcacuugucccggccuau 553hsa-miR-15b dme-miR-219 uagcagcacaucaugguuuaca 260ugauuguccaaacgcaauucuug 554 hsa-miR-23b dme-miR-276a*aucacauugccagggauuaccac 261 cagcgagguauagaguuccuacg 555 hsa-miR-27bdme-miR-276a uucacaguggcuaaguucug 262 uaggaacuucauaccgugcucu 556hsa-miR-30b dme-miR-277 uguaaacauccuacacucagc 263uaaaugcacuaucugguacgaca 557 hsa-miR-122a dme-miR-278uggagugugacaaugguguuugu 264 ucggugggacuuucguccguuu 558 hsa-miR-124adme-miR-133 uuaaggcacgcggugaaugcca 265 uugguccccuucaaccagcugu 559hsa-miR-125b dme-miR-279 ucccugagacccuaacuuguga 266ugacuagauccacacucauuaa 560 hsa-miR-128a dme-miR-33ucacagugaaccggucucuuuu 267 aggugcauuguagucgcauug 561 hsa-miR-130adme-miR-280 cagugcaauguuaaaagggc 268 uguauuuacguugcauaugaaaugaua 562hsa-miR-132 dme-miR-281-1* uaacagucuacagccauggucg 269aagagagcuguccgucgacagu 563 hsa-miR-133a dme-miR-281uugguccccuucaaccagcugu 270 ugucauggaauugcucucuuugu 564 hsa-miR-135adme-miR-282 uauggcuuuuuauuccuauguga 271 aaucuagccucuacuaggcuuugucugu 565hsa-miR-137 dme-miR-283 uauugcuuaagaauacgcguag 272 uaaauaucagcugguaauucu566 hsa-miR-138 dme-miR-284 agcugguguugugaauc 273ugaagucagcaacuugauuccagcaauug 567 hsa-miR-140 dme-miR-281-2*agugguuuuacccuaugguag 274 aagagagcuauccgucgacagu 568 hsa-miR-141dme-miR-34 aacacugucugguaaagaugg 275 uggcagugugguuagcugguug 569hsa-miR-142-5p dme-miR-124 cauaaaguagaaagcacuac 276uaaggcacgcggugaaugccaag 570 hsa-miR-142-3p dme-miR-79uguaguguuuccuacuuuaugga 277 uaaagcuagauuaccaaagcau 571 hsa-miR-143dme-miR-276b* ugagaugaagcacuguagcuca 278 cagcgagguauagaguuccuacg 572hsa-miR-144 dme-miR-276b uacaguauagaugauguacuag 279uaggaacuuaauaccgugcucu 573 hsa-miR-145 dme-miR-210guccaguuuucccaggaaucccuu 280 uugugcgugugacagcggcua 574 hsa-miR-152dme-miR-285 ucagugcaugacagaacuugg 281 uagcaccauucgaaaucagugc 575hsa-miR-153 dme-miR-100 uugcauagucacaaaaguga 282 aacccguaaauccgaacuugug576 hsa-miR-191 dme-miR-92b caacggaaucccaaaagcagcu 283aauugcacuagucccggccugc 577 hsa-miR-9 dme-miR-286 ucuuugguuaucuagcuguauga284 ugacuagaccgaacacucgugcu 578 hsa-miR-9* dme-miR-287uaaagcuagauaaccgaaagu 285 uguguugaaaaucguuugcac 579 hsa-miR-125adme-miR-87 ucccugagacccuuuaaccugug 286 uugagcaaaauuucaggugug 580hsa-miR-126* dme-miR-263b cauuauuacuuuugguacgcg 287cuuggcacugggagaauucac 581 hsa-miR-126 dme-miR-288 ucguaccgugaguaauaaugc288 uuucaugucgauuucauuucaug 582 hsa-miR-127 dme-miR-289ucggauccgucugagcuuggcu 289 uaaauauuuaaguggagccugcgacu 583 hsa-miR-129dme-bantam cuuuuugcggucugggcuugc 290 ugagaucauuuugaaagcugauu 584hsa-miR-134 dme-miR-303 ugugacugguugaccagaggg 291uuuagguuucacaggaaacuggu 585 hsa-miR-136 dme-miR-31bacuccauuuguuuugaugaugga 292 uggcaagaugucggaauagcug 586 hsa-miR-146dme-miR-304 ugagaacugaauuccauggguu 293 uaaucucaauuuguaaaugugag 587hsa-miR-149 dme-miR-305 ucuggcuccgugucuucacucc 294auuguacuucaucaggugcucug 588 hsa-miR-150 dme-miR-9cucucccaacccuuguaccagug 295 ucuuugguauucuagcuguaga 589 hsa-miR-154dme-miR-306 uagguuauccguguugccuucg 296 ucagguacuuagugacucucaa 590hsa-miR-184 dme-miR-306* uggacggagaacugauaagggu 297gggggucacucugugccugugc 591 hsa-miR-185 dme-miR-9b uggagagaaaggcaguuc 298ucuuuggugauuuuagcuguaug 592 hsa-miR-186 dme-let-7caaagaauucuccuuuugggcuu 299 ugagguaguagguuguauagu 593 hsa-miR-188dme-miR-125 caucccuugcaugguggagggu 300 ucccugagacccuaacuuguga 594hsa-miR-190 dme-miR-307 ugauauguuugauauauuaggu 301 ucacaaccuccuugagugag595 hsa-miR-193 dme-miR-308 aacuggccuacaaagucccag 302aaucacaggauuauacugugag 596 hsa-miR-194 dme-miR-31auguaacagcaacuccaugugga 303 uggcaagaugucggcauagcuga 597 hsa-miR-195dme-miR-309 uagcagcacagaaauauuggc 304 gcacuggguaaaguuuguccua 598hsa-miR-206 dme-miR-310 uggaauguaaggaagugugugg 305uauugcacacuucccggccuuu 599 hsa-miR-320 dme-miR-311aaaagcuggguugagagggcgaa 306 uauugcacauucaccggccuga 600 hsa-miR-321dme-miR-312 uaagccagggauuguggguuc 307 uauugcacuugagacggccuga 601hsa-miR-200c dme-miR-313 aauacugccggguaaugaugga 308uauugcacuuuucacagcccga 602 hsa-miR-155 dme-miR-314uuaaugcuaaucgugauagggg 309 uauucgagccaauaaguucgg 603 hsa-miR-128bdme-miR-315 ucacagugaaccggucucuuuc 310 uuuugauuguugcucagaaagc 604hsa-miR-106b dme-miR-316 uaaagugcugacagugcagau 311ugucuuuuuccgcuuacuggcg 605 hsa-miR-29c dme-miR-317uagcaccauuugaaaucgguua 312 ugaacacagcuggugguauccagu 606 hsa-miR-200adme-miR-318 uaacacugucugguaacgaugu 313 ucacugggcuuuguuuaucuca 607hsa-miR-302 dme-miR-2c uaagugcuuccauguuuugguga 314uaucacagccagcuuugaugggc 608 hsa-miR-34b dme-miR-iab-4-5paggcagugucauuagcugauug 315 acguauacugaauguauccuga 609 hsa-miR-34cdme-miR-iab-4-3p aggcaguguaguuagcugauug 316 cgguauaccuucaguauacguaac 610hsa-miR-299 ugguuuaccgucccacauacau 317 hsa-miR-301 rno-miR-322cagugcaauaguauugucaaagc 318 aaacaugaagcgcugcaaca 611 hsa-miR-99brno-miR-323 cacccguagaaccgaccuugcg 319 gcacauuacacggucgaccucu 612hsa-miR-296 rno-miR-301 agggcccccccucaauccugu 320cagugcaauaguauugucaaagcau 613 hsa-miR-130b rno-miR-324-5pcagugcaaugaugaaagggcau 321 cgcauccccuagggcauuggugu 614 hsa-miR-30erno-miR-324-3p uguaaacauccuugacugga 322 ccacugccccaggugcugcugg 615hsa-miR-340 rno-miR-325 uccgucucaguuacuuuauagcc 323ccuaguaggugcucaguaagugu 616 hsa-miR-330 rno-miR-326gcaaagcacacggccugcagaga 324 ccucugggcccuuccuccagu 617 hsa-miR-328rno-let-7d cuggcccucucugcccuuccgu 325 agagguaguagguugcauagu 618hsa-miR-342 rno-let-7d* ucucacacagaaaucgcacccguc 326cuauacgaccugcugccuuucu 619 hsa-miR-337 rno-miR-328uccagcuccuauaugaugccuuu 327 cuggcccucucugcccuuccgu 620 hsa-miR-323rno-miR-329 gcacauuacacggucgaccucu 328 aacacacccagcuaaccuuuuu 621hsa-miR-326 rno-miR-330 ccucugggcccuuccuccag 329 gcaaagcacagggccugcagaga622 hsa-miR-151 rno-miR-331 acuagacugaagcuccuugagg 330gccccugggccuauccuagaa 623 hsa-miR-135b rno-miR-333uauggcuuuucauuccuaugug 331 guggugugcuaguuacuuuu 624 hsa-miR-148brno-miR-140 ucagugcaucacagaacuuugu 332 agugguuuuacccuaugguag 625hsa-miR-331 rno-miR-140* gccccugggccuauccuagaa 333uaccacaggguagaaccacggaca 626 hsa-miR-324-5p rno-miR-336cgcauccccuagggcauuggugu 334 ucacccuuccauaucuagucu 627 hsa-miR-324-3prno-miR-337 ccacugccccaggugcugcugg 335 uucagcuccuauaugaugccuuu 628hsa-miR-338 rno-miR-148b uccagcaucagugauuuuguuga 336ucagugcaucacagaacuuugu 629 hsa-miR-339 rno-miR-338 ucccuguccuccaggagcuca337 uccagcaucagugauuuuguuga 630 hsa-miR-335 rno-miR-339ucaagagcaauaacgaaaaaugu 338 ucccuguccuccaggagcuca 631 hsa-miR-133brno-miR-341 uugguccccuucaaccagcua 339 ucgaucggucggucggucagu 632rno-miR-342 ucucacacagaaaucgcacccguc 633 osa-miR156 rno-miR-344ugacagaagagagugagcac 340 ugaucuagccaaagccugaccgu 634 osa-miR160rno-miR-345 ugccuggcucccuguaugcca 341 ugcugaccccuaguccagugc 635osa-miR162 rno-miR-346 ucgauaaaccucugcauccag 342 ugucugccugagugccugccucu636 osa-miR164 rno-miR-349 uggagaagcagggcacgugca 343cagcccugcugucuuaaccucu 637 osa-miR166 rno-miR-129 ucggaccaggcuucauucccc344 cuuuuugcggucugggcuugcu 638 osa-miR167 rno-miR-129*ugaagcugccagcaugaucua 345 aagcccuuaccccaaaaagcau 639 osa-miR169rno-miR-20 cagccaaggaugacuugccga 346 uaaagugcuuauagugcagguag 640osa-miR171 rno-miR-20* ugauugagccgcgccaauauc 347 acugcauuacgagcacuuaca641 rno-miR-350 uucacaaagcccauacacuuucac 642 rno-miR-7uggaagacuagugauuuuguu 643 rno-miR-7* caacaaaucacagucugccaua 644rno-miR-351 ucccugaggagcccuuugagccug 645 rno-miR-135buauggcuuuucauuccuaugug 646 rno-miR-151* ucgaggagcucacagucuagua 647rno-miR-151 acuagacugaggcuccuugagg 648 rno-miR-101buacaguacugugauagcugaag 649 rno-let-7a ugagguaguagguuguauaguu 650rno-let-7b ugagguaguagguugugugguu 651 rno-let-7c ugagguaguagguuguaugguu652 rno-let-7e ugagguaggagguuguauagu 653 rno-let-7fugagguaguagauuguauaguu 654 rno-let-7i ugagguaguaguuugugcu 655 rno-miR-7buggaagacuugugauuuuguu 656 rno-miR-9 ucuuugguuaucuagcuguauga 657rno-miR-10a uacccuguagauccgaauuugug 658 rno-miR-10buacccuguagaaccgaauuugu 659 rno-miR-15b uagcagcacaucaugguuuaca 660rno-miR-16 uagcagcacguaaauauuggcg 661 rno-miR-17caaagugcuuacagugcagguagu 662 rno-miR-18 uaaggugcaucuagugcagaua 663rno-miR-19b ugugcaaauccaugcaaaacuga 664 rno-miR-19augugcaaaucuaugcaaaacuga 665 rno-miR-21 uagcuuaucagacugauguuga 666rno-miR-22 aagcugccaguugaagaacugu 667 rno-miR-23a aucacauugccagggauuucc668 rno-miR-23b aucacauugccagggauuaccac 669 rno-miR-24uggcucaguucagcaggaacag 670 rno-miR-25 cauugcacuugucucggucuga 671rno-miR-26a uucaaguaauccaggauaggcu 672 rno-miR-26buucaaguaauucaggauagguu 673 rno-miR-27b uucacaguggcuaaguucug 674rno-miR-27a uucacaguggcuaaguuccgc 675 rno-miR-28 aaggagcucacagucuauugag676 rno-miR-29b uagcaccauuugaaaucagugu 677 rno-miR-29acuagcaccaucugaaaucgguu 678 rno-miR-29c uagcaccauuugaaaucgguua 679rno-miR-30c uguaaacauccuacacucucagc 680 rno-miR-30e uguaaacauccuugacugga681 rno-miR-30b uguaaacauccuacacucagc 682 rno-miR-30duguaaacauccccgacuggaag 683 rno-miR-30a cuuucagucggauguuugcagc 684rno-miR-31 aggcaagaugcuggcauagcug 685 rno-miR-32 uauugcacauuacuaaguugc686 rno-miR-33 gugcauuguaguugcauug 687 rno-miR-34buaggcaguguaauuagcugauug 688 rno-miR-34c aggcaguguaguuagcugauugc 689rno-miR-34a uggcagugucuuagcugguuguu 690 rno-miR-92 uauugcacuugucccggccug691 rno-miR-93 caaagugcuguucgugcagguag 692 rno-miR-96uuuggcacuagcacauuuuugcu 693 rno-miR-98 ugagguaguaaguuguauuguu 694rno-miR-99a aacccguagauccgaucuugug 695 rno-miR-99bcacccguagaaccgaccuugcg 696 rno-miR-100 aacccguagauccgaacuugug 697rno-miR-101 uacaguacugugauaacugaag 698 rno-miR-103agcagcauuguacagggcuauga 699 rno-miR-106b uaaagugcugacagugcagau 700rno-miR-107 agcagcauuguacagggcuauca 701 rno-miR-122auggagugugacaaugguguuugu 702 rno-miR-124a uuaaggcacgcggugaaugcca 703rno-miR-125a ucccugagacccuuuaaccugug 704 rno-miR-125bucccugagacccuaacuuguga 705 rno-miR-126* cauuauuacuuuugguacgcg 706rno-miR-126 ucguaccgugaguaauaaugc 707 rno-miR-127 ucggauccgucugagcuuggcu708 rno-miR-128a ucacagugaaccggucucuuuu 709 rno-miR-128bucacagugaaccggucucuuuc 710 rno-miR-130a cagugcaauguuaaaagggc 711rno-miR-130b cagugcaaugaugaaagggcau 712 rno-miR-132uaacagucuacagccauggucg 713 rno-miR-133a uugguccccuucaaccagcugu 714rno-miR-134 ugugacugguugaccagaggg 715 rno-miR-135auauggcuuuuuauuccuauguga 716 rno-miR-136 acuccauuuguuuugaugaugga 717rno-miR-137 uauugcuuaagaauacgcguag 718 rno-miR-138 agcugguguugugaauc 719rno-miR-139 ucuacagugcacgugucu 720 rno-miR-141 aacacugucugguaaagaugg 721rno-miR-142-5p cauaaaguagaaagcacuac 722 rno-miR-142-3puguaguguuuccuacuuuaugga 723 rno-miR-143 ugagaugaagcacuguagcuca 724rno-miR-144 uacaguauagaugauguacuag 725 rno-miR-145guccaguuuucccaggaaucccuu 726 rno-miR-146 ugagaacugaauuccauggguu 727rno-miR-150 ucucccaacccuuguaccagug 728 rno-miR-152 ucagugcaugacagaacuugg729 rno-miR-153 uugcauagucacaaaaguga 730 rno-miR-154uagguuauccguguugccuucg 731 rno-miR-181c aacauucaaccugucggugagu 732rno-miR-181a aacauucaacgcugucggugagu 733 rno-miR-181baacauucauugcugucgguggguu 734 rno-miR-183 uauggcacugguagaauucacug 735rno-miR-184 uggacggagaacugauaagggu 736 rno-miR-185 uggagagaaaggcaguuc737 rno-miR-186 caaagaauucuccuuuugggcuu 738 rno-miR-187ucgugucuuguguugcagccg 739 rno-miR-190 ugauauguuugauauauuaggu 740rno-miR-191 caacggaaucccaaaagcagcu 741 rno-miR-192 cugaccuaugaauugacagcc742 rno-miR-193 aacuggccuacaaagucccag 743 rno-miR-194uguaacagcaacuccaugugga 744 rno-miR-195 uagcagcacagaaauauuggc 745rno-miR-196 uagguaguuucauguuguugg 746 rno-miR-199acccaguguucagacuaccuguuc 747 rno-miR-200c aauacugccggguaaugaugga 748rno-miR-200a uaacacugucugguaacgaugu 749 rno-miR-200bcucuaauacugccugguaaugaug 750 rno-miR-203 gugaaauguuuaggaccacuag 751rno-miR-204 uucccuuugucauccuaugccu 752 rno-miR-205uccuucauuccaccggagucug 753 rno-miR-206 uggaauguaaggaagugugugg 754rno-miR-208 auaagacgagcaaaaagcuugu 755 rno-miR-210 cugugcgugugacagcggcug756 rno-miR-211 uucccuuugucauccuuugccu 757 rno-miR-212uaacagucuccagucacggcc 758 rno-miR-213 accaucgaccguugauuguacc 759rno-miR-214 acagcaggcacagacaggcag 760 rno-miR-216 uaaucucagcuggcaacugug761 rno-miR-217 uacugcaucaggaacugacuggau 762 rno-miR-218uugugcuugaucuaaccaugu 763 rno-miR-219 ugauuguccaaacgcaauucu 764rno-miR-221 agcuacauugucugcuggguuuc 765 rno-miR-222agcuacaucuggcuacugggucuc 766 rno-miR-223 ugucaguuugucaaauacccc 767rno-miR-290 cucaaacuaugggggcacuuuuu 768 rno-miR-291-5pcaucaaaguggaggcccucucu 769 rno-miR-291-3p aaagugcuuccacuuugugugcc 770rno-miR-292-5p acucaaacugggggcucuuuug 771 rno-miR-292-3paagugccgccagguuuugagugu 772 rno-miR-296 agggcccccccucaauccugu 773rno-miR-297 auguaugugugcauguaugcaug 774 rno-miR-298ggcagaggagggcuguucuucc 775 rno-miR-299 ugguuuaccgucccacauacau 776rno-miR-300 uaugcaagggcaagcucucuuc 777 rno-miR-320aaaagcuggguugagagggcgaa 778 rno-miR-321 uaagccagggauuguggguuc 779

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications may be made withoutdeparting from the teachings herein. The foregoing examples are providedto better illustrate the disclosed teachings and are not intended tolimit the scope of the teachings herein.

We claim:
 1. A kit comprising; a reverse transcriptase; a linker probe,wherein the linker probe comprises a stem, a loop, and a 3′target-specific portion, wherein the 3′ target specific portioncorresponds to a miRNA and is configured to be extended to form anextension reaction product complementary to the miRNA; a detector probe,wherein at least a portion of the detector probe corresponds with thestem of the linker probe, wherein the detector probe is a differentmolecule from the linker probe and wherein the detector probe comprisesa detectable label; a universal reverse primer, wherein at least aportion of the universal reverse primer corresponds with a region of theloop of the linker probe.
 2. The kit according to claim 1 furthercomprising a DNA polymerase.
 3. The kit according to claim 1 furthercomprising a primer pair.
 4. The kit according to claim 3 wherein theprimer pair comprises, a forward primer specific for the miRNA, and, theuniversal reverse primer.
 5. The kit according to claim 1 comprising aplurality of primer pairs, wherein each primer pair is in one reactionvessel of a plurality of reaction vessels.
 6. The kit according to claim1 wherein the detector probe further corresponds with at least a portionof the 3′ end region of the miRNA.
 7. A kit comprising; a reversetranscriptase; a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′ targetspecific portion corresponds to a miRNA and is configured to be extendedto form an extension reaction product complementary to the miRNA; and adetector probe, wherein at least a portion of the detector probecorresponds with the stem of the linker probe, wherein the detectorprobe is a different molecule from the linker probe and wherein thedetector probe comprises a detectable label.
 8. The kit according toclaim 7 further comprising a DNA polymerase.
 9. The kit according toclaim 7 further comprising a primer pair.
 10. The kit according to claim9 wherein the primer pair comprises, a forward primer specific for amiRNA, and, a universal reverse primer, wherein at least a portion ofthe universal reverse primer corresponds with a region of the loop ofthe linker probe.
 11. The kit according to claim 7 comprising aplurality of primer pairs, wherein each primer pair is in one reactionvessel of a plurality of reaction vessels.
 12. The kit according toclaim 7 wherein the detector probe further corresponds with at least aportion of the 3′ end region of the miRNA.
 13. The kit according toclaim 10, wherein the universal reverse primer further corresponds witha region of the stem of the linker probe.
 14. The kit according to claim10, wherein the universal reverse primer further comprises a tail regionnot corresponding to the linker probe.
 15. The kit according to claim 1,wherein the universal reverse primer further corresponds with a regionof the stem of the linker probe.
 16. The kit according to claim 1,wherein the universal reverse primer further comprises a tail region notcorresponding to the linker probe.